This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Poyart, C. Articles by Trieu-Cuot, P. Search for Related Content PubMed PubMed Citation Articles by Poyart, C. Articles by Trieu-Cuot, P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 1998, p. 41-47, Vol. 36, No. 1
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

Laboratoire Mixte Pasteur-Necker de Recherche sur les Streptocoques et Streptococcies, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France

Received 22 September 1997/Accepted 25 September 1997

	ABSTRACT

Top Abstract Introduction Materials & Methods Results & Discussion References

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA_int) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA_int fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA_int fragments on the phylogenetic tree of the sodA_int fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

	INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

The genus Streptococcus could be taxonomically divided into six major clusters which included at least 31 species (4, 8, 17, 18, 32, 34-36). These are (i) the pyogenic group, which includes S. agalactiae, S. canis, S. dysgalactiae, S. equi, S. iniae, S. porcinus, and S. pyogenes; (ii) the bovis group, which includes S. bovis, S. equinus, and S. alactolyticus; (iii) the salivarius group, which includes S. salivarius, S. thermophilus, and S. vestibularis; (iv) the mutans group, which includes S. cricetus, S. downei, S. mutans, and S. sobrinus; (v) the anginosus group (also referred to as the milleri group), which includes S. anginosus, S. constellatus, and S. intermedius; and (vi) the mitis group, which includes S. mitis, S. oralis, S. pneumoniae, S. sanguis, S. parasanguis, and S. gordonii. No single system of classification suffices for the differentiation of this heterogeneous group of organisms. Instead, classification depends on a combination of features including patterns of hemolysis observed on blood agar plates, antigenic composition, growth characteristics, biochemical reactions, and more recently, genetic analysis (3, 14, 18, 28).

In clinical laboratories, the current means of identification of streptococci rely on phenotypic tests such as those developed for the API ID 32 Strep system. However, the potential problems inherent to the use of phenotypic tests are that not all strains within a given species may be positive for a common trait (3, 18) and that the same strain may exhibit biochemical variability (15, 30). Moreover, small alterations in the realization of a test may give false results. Consequently, the routine technique based on phenotypic tests do not allow for an unequivocal identification of certain streptococcal species, in particular, those belonging to the milleri, the mutans, and the mitis groups (2, 3, 10, 18, 19). Nucleic acid-based technologies such as DNA hybridization (1, 16, 29) or amplification of selected targets (25, 27, 33) have been developed in recent years to complement and improve the identification of streptococci. We previously described a PCR assay based on the use of degenerate primers which enabled amplification of an internal fragment representing approximately 85% of the sodA gene encoding a manganese-dependent enzyme (manganese-dependent superoxide dismutase [Mn-SOD]) in various gram-positive bacteria including streptococci and enterococci (24). This gene has been identified as a target for the identification of mycobacteria at the species level by PCR (37), and we investigated in this study the sequencing of the sodA PCR product as an approach to the genotypic identification of 29 different streptococcal species including those constituting the milleri, mitis, and mutans groups.

(A report of this work was presented at the XIIIth Lancefield International Symposium [16 to 20 September 1996, Paris, France].)

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Bacterial strains and culture conditions. The main characteristics of the streptococcal strains used in this study, including the type strains, are listed in Tables 1 and 2. All strains were grown at 37°C on Columbia horse blood agar (bio-Mérieux, Marcy l'Etoile, France) in an anaerobic atmosphere. Phenotypic identifications were performed with the rapid ID 32 Strep System (API-bio-Mérieux, Marcy l'Etoile, France) according to the manufacturer's instructions. The API profiles were interpreted from the computer database for identification.

View this table: [in this window] [in a new window]   TABLE 1.   Streptococcal type strains used in this study

View this table: [in this window] [in a new window]   TABLE 2.   Comparative identification of various streptococcal strains

DNA manipulations. Rapid extraction of bacterial genomic DNA was performed as described previously (6), and primers d1 (5'-CCITAYICITAYGAYGCIYTIGARCC-3') and d2 (5'-ARRTARTAIGCRTGYTCCCAIACRTC-3') were used to amplify an internal fragment representing approximately 85% of the sodA genes of the bacterial strains. PCRs were performed with a Gene Amp System 9600 instrument (Perkin-Elmer Cetus, Roissy, France) in a final volume of 50 µl containing 250 ng of DNA as template, 0.25 µM (each) primer, 200 µM (each) deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase in a 1× amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂). The PCR mixtures were denatured (3 min at 95°C) and were then subjected to 35 cycles of amplification (90 s of annealing at 37°C, 90 s of elongation at 72°C, and 30 s of denaturation at 95°C) and to a final elongation cycle of 72°C for 10 min. The PCR products were resolved by electrophoresis on a 1% agarose gel stained with ethidium bromide.

Cloning and sequencing. Amplification products were purified on a Sephadex S-200 column (Pharmacia, Uppsala, Sweden) and were ligated into the pUC18-SmaI dephosphorylated vector by using the Sure-clone ligation kit (Pharmacia, Uppsala, Sweden). Recombinant plasmids were analyzed by colony-PCR as follows. Twelve randomly chosen clones were amplified by using the universal 21 (5'-GTAAAACGACGGCCAGT-3') and reverse (5'-AACAGCTATGACCATG-3') primers in a final volume of 50 µl containing 10³ bacteria, 0.1 µM (each) primer, 200 µM (each) deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase in a 1× amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂). The PCR mixtures were denatured (10 min at 95°C) and were then subjected to 30 cycles of amplification (90 s of annealing at 45°C, 1 min of elongation at 72°C, and 1 min of denaturation at 95°C). Colony-PCR products were directly sequenced after purification on a Sephadex S-400 column (Pharmacia). The entire nucleotide sequences of both strands of two cloned amplicons obtained from independent PCRs were determined by using the dideoxy chain termination method of Sanger with the dye primer cycle sequencing ready reaction kit on a Genetic ABI PRISM 310 Sequencer Analyzer (Perkin-Elmer, Applied Biosystem Division, Roissy, France).

Direct sequencing of the sodA_int PCR products with either of the degenerate oligonucleotides d1 and d2 was performed with the dRhodamine dye terminator sequencing kit (Perkin-Elmer, Applied Biosystem Division), as follows. After purification on a Centricon-100 Concentrator column, 200 ng of the PCR product was mixed with 8 µl of terminator reaction mixture and 10 pmol of primer in a final volume of 20 µl, and the mixture was subjected to the following thermal cycling: 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min (which was repeated for 25 cycles).

Sequence analysis. The nucleotide sequences were analyzed with Perkin-Elmer software (Sequence Analysis, Sequence Navigator, and Autoassembler). Multiple alignment of sod genes was carried out by the CLUSTAL X program (31). The construction of the unrooted phylogenetic tree was performed by the neighbor-joining method (26).

Nucleotide sequence accession numbers. The sequences were submitted to the EMBL gene bank and were assigned the accession numbers listed in Tables 1 and 2.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Amplification and sequencing of sodA_int from various streptococcal type strains. By using the primers d1 and d2 in a PCR assay, we amplified an internal fragment representing approximately 85% of the sodA gene encoding a manganese-dependent enzyme (Mn-SOD) in 29 type strains of streptococci (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. canis, S. constellatus, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). A single amplification product having the expected size of 480 bp was observed with all streptococcal species (Fig. 1 shows the results of part of this analysis). The nucleotide sequences of the sodA_int fragments from these type strains were determined following cloning into pUC18 (Table 1). Analysis of the corresponding deduced amino acid sequences (data not shown) revealed that they all possessed three histidyl residues and one aspartyl residue that supposedly serve as metal ligands at positions characteristic of Mn- or Fe-SODs (22, 23). Moreover, they all contain in the vicinity of the active site four other residues characteristic of Mn-SODs, which suggests that the corresponding enzymes are activated by Mn ions (23). We therefore concluded that the PCR products cloned and sequenced were actual sodA_int fragments. Multiple alignment of the streptococcal sodA_int sequences was carried out by the CLUSTAL X program (31), and an unrooted phylogenetic tree was constructed by the neighbor-joining method (26). Domains I and II corresponding to the degenerate oligonucleotides d1 and d2, respectively, and alignment gaps were not taken into consideration for calculations. The topology of the phylogenetic tree obtained (Fig. 2) was evaluated by bootstrap analyses to give the degree of confidence intervals for each node on the phylogenetic tree. The confidence values are given at the branches, which show possibly monophyletic clades of related organisms separated at each node. It is generally accepted that the monophyly of a clade can be accepted if the clade occurs in more than 95% of the bootstrapped trees (9). If this critical value is used, the sodA_int-based phylogenetic positions of the streptococcal species studied here were in general agreement with those inferred from an analysis of their 16S rRNA sequences (4, 17), with the following remarkable exceptions: S. agalactiae did not cluster with the species constituting the pyogenic group, and S. mutans was genetically separate from S. cricetus, S. downei, and S. sobrinus. Pairwise comparison of two given streptococcal species always revealed a lower percentage of sequence identity between their sodA_int fragments than between their 16S RNAs (Table 3 and data not shown). For example, the sequence identities of the 16S RNAs of type strains of S. mitis, S. oralis, and S. pneumoniae are greater than 99% (17), whereas those of their sodA_int fragments vary from 92% (S. mitis versus S. oralis and S. oralis versus S. pneumoniae) to 96.6% (S. mitis versus S. pneumoniae) (Table 3). These results indicate that the sodA gene might constitute a more discriminative target sequence than the 16S RNA for differentiating closely related bacterial species. On the other hand, it is worth noting that the close structural relationship (98.9% identity) observed between the sodA_int fragments of S. equi subsp. equi and S. equi subsp. zooepidemicus is consistent with their association in a single species (7).

View larger version (64K): [in this window] [in a new window]   FIG. 1.   Amplification of streptococcal type strains with the primers d1 and d2 and separation of the amplicons by 1% agarose gel electrophoresis. Lanes: 1, 1-kb ladder (Gibco, BRL); 2, S. acidominimus; 3, S. agalactiae; 4, S. alactolyticus; 5, S. anginosus; 6, S. bovis; 7, S. constellatus; 8, S. cricetus; 9, S. downei; 10, S. dysgalactiae; 11, S. equi subsp. equi; 12, S. equi subsp. zooepidemicus; 13, S. equinus; 14, S. gordonii; 15, S. intermedius; 16, S. mitis; 17, S. mutans; 18, S. oralis; 19, S. parasanguis; 20, S. pneumoniae; 21, S. porcinus; 22, S. pyogenes; 23, S. salivarius; 24, S. sanguis; 25, S. sobrinus; 26, S. thermophilus; 27, S. suis; 28, S. vestibularis. Arrowheads, 480-bp amplicon.

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Phylogenetic unrooted tree showing relationships among the sodAint fragments from various streptococcal type strains. The tree was established from an analysis of the sequences listed in Table 1 by using the neighbor-joining method (26). The value on each branch is the estimated confidence limit (expressed as a percentage) for the position of the branch as determined by bootstrap analysis. The scale bar (neighbor-joining [NJ] distance) represents 10% differences in nucleotide sequences.

View this table: [in this window] [in a new window]   TABLE 3.   Identity matrix based pairwise comparisons of sodAint fragments of streptococcal type strains

Species identification of streptococcal clinical isolates by sequencing the sodA_int gene. The design of species-specific oligonucleotides enabling the amplification of a given target DNA constitutes an interesting molecular approach for the identification of bacterial species by PCR (12, 37). The two major problems inherent to these techniques are that (i) species-specific oligonucleotides often cannot be designed for closely related species, and (ii) the number of PCRs necessary for the identification of one isolate is proportional to the number of bacterial type species that should be considered. Taking into consideration the fact that cloning and sequencing techniques are increasingly used as routine techniques in medical microbiology laboratories, we propose the sequencing of the sodA_int fragment as a molecular approach to the identification of streptococcal species. In this system, the identification of a clinical isolate is determined by the position of its sodA_int fragment on the phylogenetic tree of the sodA_int fragments of the type species (Fig. 2). To test the functionality of this approach, various typeable and nontypeable streptococcal isolates were identified by using the ID 32 Strep and/or the sodA_int systems (Table 2). We also include in this study the sequences of sodA_int fragments of known streptococcal species present in the databases. The results obtained with the sodA_int system indicated that, as expected, the two group A and the three group B streptococcal strains studied were identified as S. pyogenes and S. agalactiae, respectively. When the streptococcal strains that were unambiguously identified to the species level by the phenotypic method (API identification percentage, 99.9), the sodA_int-based identification method gave the same results (Table 2). Some discrepancies were observed for the strains the species of which were determined with an API identification percentage of less than 99.9. This was the case for NEM1164 and NEM1121, which were identified with the ID 32 Strep system as S. constellatus and S. salivarius, respectively, but which were identified with the sodA_int system as S. anginosus and S. oralis, respectively (Table 2). The sequence of the sodA_int fragment of NEM1164 displays 97 and 86% identity with the sequences of the type strains of S. anginosus and S. constellatus, respectively. The sequence of the sodA_int fragment of NEM1121 displays 96.1 and 73.6% identity with the sequences of the type strains of S. oralis and S. anginosus, respectively. On the basis of these sequence distances, we considered the sodA_int-based identification of NEM1164 (S. anginosus) and NEM1121 (S. oralis) to be more reliable than the ID 32 Strep system-based identification. Interestingly, certain strains (NEM1275, MG19, NEM666, NEM1126, and NEM895) were identified to the species level with the sodA_int system but not with ID 32 Strep system (Table 2). Finally, it is important that the intraspecies divergence between the sodA_int fragments may vary greatly depending upon the species considered, conceivably because of differences in sequence divergence rates. Consequently, it is not possible to delineate streptococcal species on the basis of the level of DNA homology. In the case of S. oralis, the levels of intraspecies divergence of the sodA_int fragments can exceed those observed between the sodA_int fragments of the type strains of S. oralis, S. mitis, and S. pneumoniae. These results might suggest that the species S. oralis, as defined, is genomically heterogeneous. Surprisingly, the sodA_int fragments from unrelated S. pneumoniae strains were found to display the highest level of intraspecies sequence identity (>99%), which suggests that transformation is not a source of sequence heterogeneity for the sodA gene, at least in pneumococci.

Concluding remarks. We have described a method that enables the reliable identification to the species level of groupable and nongroupable streptococci. It consists of (i) a PCR carried out with a single pair of degenerate oligonucleotides for amplification of a streptococcal sodA_int fragment, (ii) molecular cloning of the resulting amplicon into an Escherichia coli vector, and (iii) sequencing of the insert on both DNA strands. Sequencing of a streptococcal sodA_int fragment by using this procedure necessitates 72 h following receipt of the bacterial strain; however, we anticipate further simplification and/or automation of various steps of this method. For example, based on the observation that a single abundant PCR product was obtained by using the degenerate sod primers, whatever the species tested (Fig. 1), we successfully tried to sequence directly both strands of the amplified DNA with either of the degenerate PCR primers (Fig. 3 shows part of the results of that analysis). Removal of the cloning step greatly enhances the applicability of the procedure and reduces the delay required for bacterial identification to 24 h. This method might be useful in reference laboratories for the characterization strains that could not be assigned to a species on the basis of their conventional phenotypic reaction. It provides a convenient alternative to the DNA-DNA hybridization method, which constitutes the reference technique for the identification of strains to the species level. We are expanding the applicability of this approach by determining the nucleotide sequence of sodA_int fragments from other species of streptococci as well as from other related genera such as Abiotrophia, Enterococcus, Gemella, Leuconostoc, and Pediococcus.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   Electropherograms showing part of the nucleotide sequence of sodAint from S. porcinus. Sequence reactions were carried out with a pUC18sodAint recombinant plasmid with the 21 dye primer sequencing kit (A) and sodAint PCR product with the degenerate oligonucleotide d2 and the dRhodamine dye terminator sequencing kit (B).

	ACKNOWLEDGMENTS

We thank C. Bizet for the gift of streptococcal type strains (CIP); A. Buu-Hoï, L. Gutman, N. Fortineau, and O. Gaillot for gifts of clinical isolates; and E. Abachin for technical assistance.

This work was supported by the Institut Pasteur and by the University Paris V.

	FOOTNOTES

^* Corresponding author. Mailing address: Laboratoire Mixte Pasteur-Necker de Recherche sur les Streptocoques et Streptococcies, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France. Phone: (33) (1) 40 61 56 79. Fax: (33) (1) 40 61 55 92. E-mail: ptrieu{at}pasteur.fr.

	REFERENCES

Top Abstract Introduction Materials & Methods Results & Discussion References

1.	Adnan, S., N. Li, H. Miura, Y. Hashimoto, H. Yamamoto, and T. Ezaki. 1993. Covalently immobilized DNA plate for luminometric DNA-DNA hybridization to identify viridans streptococci in under 2 hours. FEMS Microbiol. Lett. 106:139-142[Medline].
2.	Ahmet, Z., M. Warren, and E. T. Houang. 1995. Species identification of members of the Streptococcus milleri group isolated from the vagina by ID 32 Strep system and differential phenotypic characteristics. J. Clin. Microbiol. 33:1592-1595[Abstract].
3.	Beighton, D., J. M. Hardie, and A. Whiley. 1991. A scheme for the identification of viridans streptococci. J. Med. Microbiol. 35:367-372[Abstract].
4.	Bentley, R. W., J. A. Leigh, and M. D. Collins. 1991. Intrageneric structure of Streptococcus based on comparative analysis of small-subunit rRNA sequences. Int. J. Syst. Bacteriol. 41:487-494[Abstract/Free Full Text].
5.	Clermont, D., and T. Horaud. 1990. Identification of chromosomal antibiotic resistance genes in Streptococcus anginosus ("S. milleri"). Antimicrob. Agents Chemother. 34:1685-1690[Abstract/Free Full Text].
6.	de Lamballerie, X., C. Zandotti, C. Vignoli, C. Bollet, and P. de Micco. 1992. A one-step microbial DNA extraction method using "Chelex 100" suitable for gene amplification. Res. Microbiol. 143:785-790[Medline].
7.	Farrow, J. A. E., and M. D. Collins. 1984. Taxonomic studies on streptococci of serological groups C, G, and L and possibly related taxa. Syst. Appl. Microbiol. 5:483-493.
8.	Farrow, J. A. E., J. Kruze, B. A. Phillips, A. J. Bramley, and M. D. Collins. 1984. Taxonomic studies on Streptococcus bovis and Streptococcus equinus: description of Streptococcus alactolyticus sp. nov. Syst. Appl. Microbiol. 5:467-482.
9.	Felsenstein, J. 1985. Confidence limits on phylogeny and approach using the boostrap. Evolution 39:783-791.
10.	Flynn, C. E., and K. L. Ruoff. 1995. Identification of Streptococcus milleri group isolates to the species level with a commercially available rapid test. J. Clin. Microbiol. 33:2704-2706[Abstract].
11.	Gaillot, O., C. Poyart, P. Berche, and P. Trieu-Cuot. Molecular characterization and expression analysis of the superoxide dismutase gene from Streptococcus agalactiae. Gene, in press.
12.	Garnier, F., G. Gerbaud, P. Courvalin, and M. Galimand. 1997. Identification of clinically relevant viridans group streptococci to the species level by PCR. J. Clin. Microbiol. 35:2337-2341[Abstract].
13.	Gibson, C. M., and M. G. Caparon. 1996. Insertional inactivation of Streptococcus pyogenes sod suggests that prtF is regulated in response to a superoxide signal. J. Bacteriol. 178:4688-4695[Abstract/Free Full Text].
14.	Hardie, J. M. 1986. Genus Streptococcus, p. 1043-1071. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology. The Williams & Wilkins Co, Baltimore, Md.
15.	Hillman, J. D., S. W. Andrews, S. Painetr, and P. Stashenko. 1989. Adaptative changes in a strain of Streptococcus mutans during colonization of the human oral cavity. Microb. Ecol. Health Dis. 2:231-239.
16.	Jacobs, J. A., C. S. Schot, A. E. Bunschoten, and L. M. Schouls. 1996. Rapid species identification of "Streptococcus milleri" strains by line blot hybridization: identification of a distinct 16S rRNA population closely related to Streptococcus constellatus. J. Clin. Microbiol. 34:1717-1721[Abstract].
17.	Kawamura, Y., X.-G. Hou, F. Sultana, H. Miura, and T. Ezaki. 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/Free Full Text].
18.	Kilian, M., L. Mikkelsen, and J. Henrichsen. 1989. Taxonomic studies 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.
19.	Kilpper-Bälz, R., B. L. Williams, R. Lutticken, and K. H. Schleifer. 1984. Relatedness of `Streptococcus miller' with Streptococcus anginosus an Streptococcus constellatus. Syst. Appl. Microbiol. 5:494-500.
20.	Le Bouguénec, C., T. Horaud, G. Bieth, R. Colimon, and C. Dauguet. 1984. Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids. Mol. Gen. Genet. 194:377-387[Medline].
21.	Nakayama, K. 1992. Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants. J. Bacteriol. 174:4928-4934[Abstract/Free Full Text].
22.	Parker, M. W., and C. C. F. Balke. 1988. Crystal structure of manganese superoxide dismutase from Bacillus stearothermophilus at 2.4 Å resolution. J. Mol. Biol. 199:649-661[Medline].
23.	Parker, M. W., and C. C. F. Blake. 1988. Iron- and manganese-containing superoxide dismutases can be distinguished by analysis of their primary structures. FEBS Lett. 229:377-382[Medline].
24.	Poyart, C., P. Berche, and P. Trieu-Cuot. 1995. Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers. FEMS Microbiol. Lett. 131:41-45[Medline].
25.	Rudney, J. D., and C. J. Larson. 1994. Use of restriction fragment polymorphism analysis of rRNA genes to assign species to unknown clinical isolates of oral viridans streptococci. J. Clin. Microbiol. 32:437-443[Abstract/Free Full Text].
26.	Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract].
27.	Saruta, K., T. Matsunaga, S. Hoshina, M. Kono, S. Kitahara, S. Kanemoto, O. Sakai, and K. Machida. 1995. Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region. FEMS Microbiol. Lett. 132:165-170[Medline].
28.	Schleifer, K. H., and R. Kilpper-Bälz. 1987. Molecular and chemotaxonomic approaches to the classification of streptococci, enterococci, and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
29.	Schmidhuber, S., W. Ludwig, and K. H. Schleifer. 1988. Construction of a DNA probe for the specific identification of Streptococcus oralis. J. Clin. Microbiol. 26:1042-1044[Abstract/Free Full Text].
30.	Tardif, G., M. C. Sulavik, G. W. Jones, and D. B. Clewell. 1989. Spontaneous switching of the sucrose-promoted colony phenotype in Streptococcus sanguis. Infect. Immun. 57:3945-3948[Abstract/Free Full Text].
31.	Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680[Abstract/Free Full Text].
32.	Whiley, R. A., and D. Beighton. 1991. Emended descriptions and recognition of Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus as distinct species. Int. J. Syst. Bacteriol. 41:1-5[Abstract/Free Full Text].
33.	Whiley, R. A., B. Duke, J. M. Hardie, and L. M. C. Hall. 1995. Heterogeneity among 16S-23S rRNA intergenic spacers of species within the `Streptococcus milleri group.' Microbiology 141:1461-1467[Abstract].
34.	Whiley, R. A., H. Y. Fraser, C. W. I. Douglas, J. M. Hardie, A. M. Williams, and M. D. Collins. 1990. Streptococcus parasanguis sp. nov., an atypical viridans Streptococcus from human clinical specimens. FEMS Microbiol. Lett. 68:115-122.
35.	Whiley, R. A., and J. M. Hardie. 1988. Streptococcus vestibularis sp. nov. from the human oral cavity. Int. J. Syst. Bacteriol. 38:335-339[Abstract/Free Full Text].
36.	Whiley, R. A., R. R. B. Russell, J. M. Hardie, and D. Beighton. 1988. Streptococcus downeii sp. nov. for strains previously described as Streptococcus mutans serotype H. Int. J. Syst. Bacteriol. 38:25-29.
37.	Zolg, J. W., and S. Philippi-Schulz. 1994. The superoxide dismutase gene, a target for detection and identification of mycobacteria by PCR. J. Clin. Microbiol. 32:2801-2812[Abstract/Free Full Text].

This article has been cited by other articles:

• Cortes, P. R., Orio, A. G. A., Regueira, M., Pinas, G. E., Echenique, J. (2008). Characterization of In Vitro-Generated and Clinical Optochin-Resistant Strains of Streptococcus pneumoniae Isolated from Argentina. J. Clin. Microbiol. 46: 1930-1934 [Abstract] [Full Text]  
• Chen, H.-J., Tsai, J.-C., Chang, T.-C., Hung, W.-C., Tseng, S.-P., Hsueh, P.-R., Teng, L.-J. (2008). PCR-RFLP assay for species and subspecies differentiation of the Streptococcus bovis group based on groESL sequences. J Med Microbiol 57: 432-438 [Abstract] [Full Text]  
• Faibis, F., Mihaila, L., Perna, S., Lefort, J.-F., Demachy, M.-C., Le Fleche-Mateos, A., Bouvet, A. (2008). Streptococcus sinensis: an emerging agent of infective endocarditis. J Med Microbiol 57: 528-531 [Abstract] [Full Text]  
• Milinovich, G. J., Burrell, P. C., Pollitt, C. C., Bouvet, A., Trott, D. J. (2008). Streptococcus henryi sp. nov. and Streptococcus caballi sp. nov., isolated from the hindgut of horses with oligofructose-induced laminitis. Int. J. Syst. Evol. Microbiol. 58: 262-266 [Abstract] [Full Text]  
• Warburton, P. J., Palmer, R. M., Munson, M. A., Wade, W. G. (2007). Demonstration of in vivo transfer of doxycycline resistance mediated by a novel transposon. J Antimicrob Chemother 60: 973-980 [Abstract] [Full Text]  
• Sun, J.-R., Yan, J.-C., Yeh, C.-Y., Lee, S.-Y., Lu, J.-J. (2007). Invasive infection with Streptococcus iniae in Taiwan. J Med Microbiol 56: 1246-1249 [Abstract] [Full Text]  
• Ip, M., Chau, S. S. L., Chi, F., Tang, J., Chan, P. K. (2007). Fluoroquinolone Resistance in Atypical Pneumococci and Oral Streptococci: Evidence of Horizontal Gene Transfer of Fluoroquinolone Resistance Determinants from Streptococcus pneumoniae. Antimicrob. Agents Chemother. 51: 2690-2700 [Abstract] [Full Text]  
• Friedrichs, C., Rodloff, A. C., Chhatwal, G. S., Schellenberger, W., Eschrich, K. (2007). Rapid Identification of Viridans Streptococci by Mass Spectrometric Discrimination. J. Clin. Microbiol. 45: 2392-2397 [Abstract] [Full Text]  
• Davies, M. R., McMillan, D. J., Van Domselaar, G. H., Jones, M. K., Sriprakash, K. S. (2007). Phage 3396 from a Streptococcus dysgalactiae subsp. equisimilis Pathovar May Have Its Origins in Streptococcus pyogenes. J. Bacteriol. 189: 2646-2652 [Abstract] [Full Text]  
• Tung, S. K., Teng, L. J., Vaneechoutte, M., Chen, H. M., Chang, T. C. (2007). Identification of species of Abiotrophia, Enterococcus, Granulicatella and Streptococcus by sequence analysis of the ribosomal 16S-23S intergenic spacer region. J Med Microbiol 56: 504-513 [Abstract] [Full Text]  
• Haanpera, M., Jalava, J., Huovinen, P., Meurman, O., Rantakokko-Jalava, K. (2007). Identification of Alpha-Hemolytic Streptococci by Pyrosequencing the 16S rRNA Gene and by Use of VITEK 2. J. Clin. Microbiol. 45: 762-770 [Abstract] [Full Text]  
• Delorme, C., Poyart, C., Ehrlich, S. D., Renault, P. (2007). Extent of Horizontal Gene Transfer in Evolution of Streptococci of the Salivarius Group. J. Bacteriol. 189: 1330-1341 [Abstract] [Full Text]  
• Gatson, J. W., Benz, B. F., Chandrasekaran, C., Satomi, M., Venkateswaran, K., Hart, M. E. (2006). Bacillus tequilensis sp. nov., isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis.. Int. J. Syst. Evol. Microbiol. 56: 1475-1484 [Abstract] [Full Text]  
• Glazunova, O. O., Raoult, D., Roux, V. (2006). Streptococcus massiliensis sp. nov., isolated from a patient blood culture.. Int. J. Syst. Evol. Microbiol. 56: 1127-1131 [Abstract] [Full Text]  
• Innings, A., Krabbe, M., Ullberg, M., Herrmann, B. (2005). Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene. J. Clin. Microbiol. 43: 5983-5991 [Abstract] [Full Text]  
• Hoshino, T., Fujiwara, T., Kilian, M. (2005). Use of Phylogenetic and Phenotypic Analyses To Identify Nonhemolytic Streptococci Isolated from Bacteremic Patients. J. Clin. Microbiol. 43: 6073-6085 [Abstract] [Full Text]  
• Sangvik, M., Littauer, P., Simonsen, G. S., Sundsfjord, A., Dahl, K. H. (2005). mef(A), mef(E) and a new mef allele in macrolide-resistant Streptococcus spp. isolates from Norway. J Antimicrob Chemother 56: 841-846 [Abstract] [Full Text]  
• Poirel, L., Brinas, L., Verlinde, A., Ide, L., Nordmann, P. (2005). BEL-1, a Novel Clavulanic Acid-Inhibited Extended-Spectrum {beta}-Lactamase, and the Class 1 Integron In120 in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 49: 3743-3748 [Abstract] [Full Text]  
• Daley, P., Church, D. L., Gregson, D. B., Elsayed, S. (2005). Species-Level Molecular Identification of Invasive "Streptococcus milleri" Group Clinical Isolates by Nucleic Acid Sequencing in a Centralized Regional Microbiology Laboratory. J. Clin. Microbiol. 43: 2987-2988 [Abstract] [Full Text]  
• Littauer, P., Sangvik, M., Caugant, D. A., Hoiby, E. A., Simonsen, G. S., Sundsfjord, A., the Norwegian Macrolide Study Group, (2005). Molecular Epidemiology of Macrolide-Resistant Isolates of Streptococcus pneumoniae Collected from Blood and Respiratory Specimens in Norway. J. Clin. Microbiol. 43: 2125-2132 [Abstract] [Full Text]  
• Gautier, A.-L., Dubois, D., Escande, F., Avril, J.-L., Trieu-Cuot, P., Gaillot, O. (2005). Rapid and Accurate Identification of Human Isolates of Pasteurella and Related Species by Sequencing the sodA Gene. J. Clin. Microbiol. 43: 2307-2314 [Abstract] [Full Text]  
• Leclercq, R., Huet, C., Picherot, M., Trieu-Cuot, P., Poyart, C. (2005). Genetic Basis of Antibiotic Resistance in Clinical Isolates of Streptococcus gallolyticus (Streptococcus bovis). Antimicrob. Agents Chemother. 49: 1646-1648 [Abstract] [Full Text]  
• Chen, C. C., Teng, L. J., Kaiung, S., Chang, T. C. (2005). Identification of Clinically Relevant Viridans Streptococci by an Oligonucleotide Array. J. Clin. Microbiol. 43: 1515-1521 [Abstract] [Full Text]  
• Hassan, A. A., Akineden, O., Usleber, E. (2005). Identification of Streptococcus canis Isolated from Milk of Dairy Cows with Subclinical Mastitis. J. Clin. Microbiol. 43: 1234-1238 [Abstract] [Full Text]  
• Arbique, J. C., Poyart, C., Trieu-Cuot, P., Quesne, G., Carvalho, M. d. G. S., Steigerwalt, A. G., Morey, R. E., Jackson, D., Davidson, R. J., Facklam, R. R. (2004). Accuracy of Phenotypic and Genotypic Testing for Identification of Streptococcus pneumoniae and Description of Streptococcus pseudopneumoniae sp. nov.. J. Clin. Microbiol. 42: 4686-4696 [Abstract] [Full Text]  
• Jackson, C. R., Fedorka-Cray, P. J., Barrett, J. B. (2004). Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci. J. Clin. Microbiol. 42: 3558-3565 [Abstract] [Full Text]  
• Picard, F. J., Ke, D., Boudreau, D. K., Boissinot, M., Huletsky, A., Richard, D., Ouellette, M., Roy, P. H., Bergeron, M. G. (2004). Use of tuf Sequences for Genus-Specific PCR Detection and Phylogenetic Analysis of 28 Streptococcal Species. J. Clin. Microbiol. 42: 3686-3695 [Abstract] [Full Text]  
• Chen, C. C., Teng, L. J., Chang, T. C. (2004). Identification of Clinically Relevant Viridans Group Streptococci by Sequence Analysis of the 16S-23S Ribosomal DNA Spacer Region. J. Clin. Microbiol. 42: 2651-2657 [Abstract] [Full Text]  
• Bosshard, P. P., Abels, S., Altwegg, M., Bottger, E. C., Zbinden, R. (2004). Comparison of Conventional and Molecular Methods for Identification of Aerobic Catalase-Negative Gram-Positive Cocci in the Clinical Laboratory. J. Clin. Microbiol. 42: 2065-2073 [Abstract] [Full Text]  
• Drancourt, M., Roux, V., Fournier, P.-E., Raoult, D. (2004). rpoB Gene Sequence-Based Identification of Aerobic Gram-Positive Cocci of the Genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella. J. Clin. Microbiol. 42: 497-504 [Abstract] [Full Text]  
• Coenye, T., Vandamme, P. (2003). Extracting phylogenetic information from whole-genome sequencing projects: the lactic acid bacteria as a test case. Microbiology 149: 3507-3517 [Abstract] [Full Text]  
• Tapp, J., Thollesson, M., Herrmann, B. (2003). Phylogenetic relationships and genotyping of the genus Streptococcus by sequence determination of the RNase P RNA gene, rnpB. Int. J. Syst. Evol. Microbiol. 53: 1861-1871 [Abstract] [Full Text]  
• Martin-Galiano, A. J., Balsalobre, L., Fenoll, A., de la Campa, A. G. (2003). Genetic Characterization of Optochin-Susceptible Viridans Group Streptococci. Antimicrob. Agents Chemother. 47: 3187-3194 [Abstract] [Full Text]  
• Dahl, K. H., Sundsfjord, A. (2003). Transferable vanB2 Tn5382-Containing Elements in Fecal Streptococcal Strains from Veal Calves. Antimicrob. Agents Chemother. 47: 2579-2583 [Abstract] [Full Text]  
• Chang, Y.-H., Shangkuan, Y.-H., Lin, H.-C., Liu, H.-W. (2003). PCR Assay of the groEL Gene for Detection and Differentiation of Bacillus cereus Group Cells. Appl. Environ. Microbiol. 69: 4502-4510 [Abstract] [Full Text]  
• Gilmore, K. S., Srinivas, P., Akins, D. R., Hatter, K. L., Gilmore, M. S. (2003). Growth, Development, and Gene Expression in a Persistent Streptococcus gordonii Biofilm. Infect. Immun. 71: 4759-4766 [Abstract] [Full Text]  
• Li, Y., Pan, Y., Qi, F., Caufield, P. W. (2003). Identification of Streptococcus sanguinis with a PCR-Generated Species-Specific DNA Probe. J. Clin. Microbiol. 41: 3481-3486 [Abstract] [Full Text]  
• Schlegel, L., Grimont, F., Grimont, P. A. D., Bouvet, A. (2003). Identification of Major Streptococcal Species by rrn-Amplified Ribosomal DNA Restriction Analysis. J. Clin. Microbiol. 41: 657-666 [Abstract] [Full Text]  
• Facklam, R. (2002). What Happened to the Streptococci: Overview of Taxonomic and Nomenclature Changes. Clin. Microbiol. Rev. 15: 613-630 [Abstract] [Full Text]  
• Jakubovics, N. S., Smith, A. W., Jenkinson, H. F. (2002). Oxidative stress tolerance is manganese (Mn2+) regulated in Streptococcus gordonii. Microbiology 148: 3255-3263 [Abstract] [Full Text]  
• Teng, L.-J., Hsueh, P.-R., Tsai, J.-C., Chen, P.-W., Hsu, J.-C., Lai, H.-C., Lee, C.-N., Ho, S.-W. (2002). groESL Sequence Determination, Phylogenetic Analysis, and Species Differentiation for Viridans Group Streptococci. J. Clin. Microbiol. 40: 3172-3178 [Abstract] [Full Text]  
• Obregon, V., Garcia, P., Garcia, E., Fenoll, A., Lopez, R., Garcia, J. L. (2002). Molecular Peculiarities of the lytA Gene Isolated from Clinical Pneumococcal Strains That Are Bile Insoluble. J. Clin. Microbiol. 40: 2545-2554 [Abstract] [Full Text]  
• Vaillancourt, K., Moineau, S., Frenette, M., Lessard, C., Vadeboncoeur, C. (2002). Galactose and Lactose Genes from the Galactose-Positive Bacterium Streptococcus salivarius and the Phylogenetically Related Galactose-Negative Bacterium Streptococcus thermophilus: Organization, Sequence, Transcription, and Activity of the gal Gene Products. J. Bacteriol. 184: 785-793 [Abstract] [Full Text]  
• Teng, L.-J., Hsueh, P.-R., Ho, S.-W., Luh, K.-T. (2001). High Prevalence of Inducible Erythromycin Resistance among Streptococcus bovis Isolates in Taiwan. Antimicrob. Agents Chemother. 45: 3362-3365 [Abstract] [Full Text]  
• Poyart, C., Quesne, G., Boumaila, C., Trieu-Cuot, P. (2001). Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the sodA Gene as a Target. J. Clin. Microbiol. 39: 4296-4301 [Abstract] [Full Text]  
• Whatmore, A. M., Engler, K. H., Gudmundsdottir, G., Efstratiou, A. (2001). Identification of Isolates of Streptococcus canis Infecting Humans. J. Clin. Microbiol. 39: 4196-4199 [Abstract] [Full Text]  
• Hedegaard, J., Okkels, H., Bruun, B., Kilian, M., Mortensen, K. K., Norskov-Lauritsen, N. (2001). Phylogeny of the genus Haemophilus as determined by comparison of partial infB sequences. Microbiology 147: 2599-2609 [Abstract] [Full Text]  
• Idigoras, P., Valiente, A., Iglesias, L., Trieu-Cuot, P., Poyart, C. (2001). Meningitis Due to Streptococcus salivarius. J. Clin. Microbiol. 39: 3017-3017 [Full Text]  
• Amoroso, A., Demares, D., Mollerach, M., Gutkind, G., Coyette, J. (2001). All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level {beta}-Lactam-Resistant Clinical Isolate of Streptococcus mitis. Antimicrob. Agents Chemother. 45: 2075-2081 [Abstract] [Full Text]  
• Baele, M., Storms, V., Haesebrouck, F., Devriese, L. A., Gillis, M., Verschraegen, G., de Baere, T., Vaneechoutte, M. (2001). Application and Evaluation of the Interlaboratory Reproducibility of tRNA Intergenic Length Polymorphism Analysis (tDNA-PCR) for Identification of Streptococcus Species. J. Clin. Microbiol. 39: 1436-1442 [Abstract] [Full Text]  
• Guerin, F., Varon, E., Hoï, A. B., Gutmann, L., Podglajen, I. (2000). Fluoroquinolone Resistance Associated with Target Mutations and Active Efflux in Oropharyngeal Colonizing Isolates of Viridans Group Streptococci. Antimicrob. Agents Chemother. 44: 2197-2200 [Abstract] [Full Text]  
• Caufield, P. W., Dasanayake, A. P., Li, Y., Pan, Y., Hsu, J., Hardin, J. M. (2000). Natural History of Streptococcus sanguinis in the Oral Cavity of Infants: Evidence for a Discrete Window of Infectivity. Infect. Immun. 68: 4018-4023 [Abstract] [Full Text]  
• Poyart, C., Quesne, G., Acar, P., Berche, P., Trieu-Cuot, P. (2000). Characterization of the Tn916-like Transposon Tn3872 in a Strain of Abiotrophia defectiva (Streptococcus defectivus) Causing Sequential Episodes of Endocarditis in a Child. Antimicrob. Agents Chemother. 44: 790-793 [Abstract] [Full Text]  
• Majewski, J., Zawadzki, P., Pickerill, P., Cohan, F. M., Dowson, C. G. (2000). Barriers to Genetic Exchange between Bacterial Species: Streptococcus pneumoniae Transformation. J. Bacteriol. 182: 1016-1023 [Abstract] [Full Text]  
• Poyart, C., Quesnes, G., Trieu-Cuot, P. (2000). Sequencing the Gene Encoding Manganese-Dependent Superoxide Dismutase for Rapid Species Identification of Enterococci. J. Clin. Microbiol. 38: 415-418 [Abstract] [Full Text]  
• Brown, A. E., Rogers, J. D., Haase, E. M., Zelasko, P. M., Scannapieco, F. A. (1999). Prevalence of the Amylase-Binding Protein A Gene (abpA) in Oral Streptococci. J. Clin. Microbiol. 37: 4081-4085 [Abstract] [Full Text]  
• Alam, S., Brailsford, S. R., Whiley, R. A., Beighton, D. (1999). PCR-Based Methods for Genotyping Viridans Group Streptococci. J. Clin. Microbiol. 37: 2772-2776 [Abstract] [Full Text]  
• Kawamura, Y., Whiley, R. A., Shu, S.-E., Ezaki, T., Hardie, J. M. (1999). Genetic approaches to the identification of the mitis group within the genus Streptococcus. Microbiology 145: 2605-2613 [Abstract] [Full Text]  
• Ratcliff, R. M., Lanser, J. A., Manning, P. A., Heuzenroeder, M. W. (1998). Sequence-Based Classification Scheme for the Genus Legionella Targeting the mip Gene. J. Clin. Microbiol. 36: 1560-1567 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Poyart, C. Articles by Trieu-Cuot, P. Search for Related Content PubMed PubMed Citation Articles by Poyart, C. Articles by Trieu-Cuot, P.