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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1998 Jan;36(1):41–47. doi: 10.1128/jcm.36.1.41-47.1998

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

Claire Poyart 1, Gilles Quesne 1, Stephane Coulon 1, Patrick Berche 1, Patrick Trieu-Cuot 1,*
PMCID: PMC124804  PMID: 9431917

Abstract

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint) 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 sodAint 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 sodAint fragments on the phylogenetic tree of the sodAint 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.

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

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.

TABLE 1.

Streptococcal type strains used in this study

Straina Other designationb Relevant characteristics sodAint accession no.
S. acidominimus CIP 82.4T NCDO 2025 Type strain Z95892
S. agalactiae CIP 103227T ATCC 13813 Type strain Z95893
S. alactolyticus CIP 103244T ATCC 43077 Type strain Z95894
S. anginosus CIP 102921T ATCC 33397 Type strain Z95895
S. bovis CIP 102302T ATCC 33317 Type strain Z95896
S. canis CIP 103223T ATCC43496 Type strain Z99175
S. constellatus CIP 103247T ATCC 27823 Type strain Z95897
S. cricetus CIP 102510T ATCC 19642 Type strain Z95898
S. downei CIP 103222T ATCC 33748 Type strain Z95899
S. dysgalactiae CIP 102914T ATCC 43078 Type strain Z95900
S. equinus CIP 102504T ATCC 9812 Type strain Z95903
S. equi subsp. equi CIP 102910T ATCC 33398 Type strain Z95901
S. equi subsp. zooepidemicus CIP 103228T ATCC 43079 Type strain Z95902
S. gordonii CIP 105258T ATCC 10558 Type strain Z95905
S. iniae CIP 102508T ATCC 29178 Type strain Z99176
S. intermedius CIP 103248T ATCC 27335 Type strain Z95908
S. mitis CIP 103335T NCTC 12261 Type strain Z95909
S. mutans CIP 103694 ATCC 35668 Quality control strain for API product Z95910
S. oralis CIP 10922T ATCC 35037 Type strain Z95911
S. parasanguis CIP 104372T ATCC 15910 Type strain Z95913
S. pneumoniae CIP 102911T ATCC 33400 Type strain Z95914
S. porcinus CIP 103218T ATCC 43138 Type strain Z99177
S. pyogenes CIP 56.41T ATCC 12344 Type strain Z95915
S. salivarius CIP 102509T ATCC 19645 Type strain Z95916
S. sanguis CIP 55.1328T ATCC 10556 Type strain Z95918
S. sobrinus CIP 103230T ATCC 33478 Type strain Z95919
S. suis CIP 103217T ATCC 43765 Type strain Z95920
S. thermophilus CIP 102303T ATCC 19258 Type strain Z95921
S. vestibularis CIP 103363T ATCC 49124 Type strain Z95922
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a

CIP, Collection de l’Institut Pasteur. 

b

NCDO, National Collection of Dairy Organisms; ATCC, American Type Culture Collection; NCTC, National Collection of Type Cultures. 

TABLE 2.

Comparative identification of various streptococcal strains

Straina Relevant characteristicsb Species identification
sodAint accession no.
ID 32 Strepc sodAintd
S. agalactiae
 CIP 82.45 Lancefield group B (ATCC 12403) ND S. agalactiae (98.6) Z99178
 NEM318 Lancefield group B ND S. agalactiae (97.9) Z99179
 NEM1317 Lancefield group B ND S. agalactiae (98.4) Z99180
Milleri group
 NEM1164 Nontypeable, β-hemolytic S. constellatus (99.6) S. anginosus (97) Z99181
 NEM1166 Nontypeable, β-hemolytic S. anginosus (99.8) S. anginosus (97.4) Z99182
 NEM1124 Lancefield group F, β-hemolytic S. constellatus (99.9) S. constellatus (98.1) Z99183
 NEM1162 Lancefield group F, β-hemolytic S. constellatus (99.6) S. constellatus (99.1) Z99184
 NEM1165 Nontypeable S. constellatus (93.1) S. constellatus (98.9) Z99185
 NEM1275 Nontypeable S. constellatus (63.9) S. constellatus (99.1) Z99186
 MG19 Nontypeable Milleri group S. constellatus (99.1) Z99187
Mitis group
 CIP 103221 ATCC 33399 S. gordonii (?) S. gordonii (95.9) Z99189
 BM120 Strain Challis S. gordonii (99.9) S. gordonii (98.2) Z99188
 NEM666 Nontypeable ? S. gordonii (95.9) Z99190
 NEM1222 Nontypeable S. mitis (95.6) S. mitis (97.8) Z99192
 NEM1126 Lancefield group C S. mitis (59.5) S. mitis (97.8) Z99191
S. mutans
 GS-5 ND S. mutans (99.8) D01037
 NEM1163 Nontypeable S. mutans (99.9) S. mutans (99.5) Z99193
S. oralis
 CIP 103216 ATCC 10557 S. oralis (93.7) Z99194
 NEM1121 Lancefield group C S. salivarius (99.8) S. oralis (96.1) Z99195
 NEM895 Nontypeable ? S. parasanguis (97.8) Z99196
S. pneumoniae
 NEM667 Serotype 11 ND S. pneumoniae (100) Z99246
 NEM1278 Serotype 23F ND S. pneumoniae (99.8) Z99204
 NEM1279 Serotype 23F, Penr ND S. pneumoniae (100) Z99205
 NEM1280 Serotype 23F, Penr ND S. pneumoniae (100) Z99206
 NEM1251 Serotype 16 ND S. pneumoniae (100) Z99201
 NEM1252 Serotype 18 ND S. pneumoniae (100) Z99202
 NEM1253 Serotype 6 ND S. pneumoniae (100) Z99203
 NEM1122 Serotype 23F, Optr ? S. pneumoniae (99.8) Z99200
S. pyogenes
 BM105 Lancefield group A ND S. pyogenes (100) Z49247
 HSC5 Lancefield group A ND S. pyogenes (100) U43776
S. salivarius
 CIP 102505 ATCC 13419 S. salivarius (?) S. salivarius (96.3) Z99197
 NEM1250 Lancefield group D S. salivarius (99.9) S. salivarius (98.6) Z99198
 NEM1257 Nontypeable S. salivarius (99.5) S. salivarius (96.3) Z99199
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a

Data for all strains except S. agalactiae CIP 82.45 (24), S. agalactiae NEM318 (11), MG19 (5), S. gordonii BM120 (20), S. mutans GS-5 (21), S. pneumoniae NEM667 (24), S. pyogenes BM105 (24), and S. pyogenes HSC5 (13) are from this work. 

b

Optor and Penr, resistance to optochin and penicillin, respectively. 

c

The rapid ID 32 Strep system (API-bio-Mérieux) was used according to the manufacturer’s instructions to identify isolates to the species level. The API profiles were interpreted from the computer database for identification. The numbers in parentheses indicate the API identification percentage. ND, not determined; ?, undetermined. 

d

The species identification was based on the phylogenic position of the sodAint fragment of the strain studied relative to those of the type strains, as indicated in Fig. 1. The numbers in parentheses indicate the percent identity of the sodAint fragment with that of the corresponding type strain. 

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 sodAint 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

Amplification and sequencing of sodAint 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 sodAint 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 sodAint fragments. Multiple alignment of the streptococcal sodAint 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 sodAint-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 sodAint 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 sodAint 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 sodAint fragments of S. equi subsp. equi and S. equi subsp. zooepidemicus is consistent with their association in a single species (7).

FIG. 1.

FIG. 1

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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.

FIG. 2.

FIG. 2

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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.

TABLE 3.

Identity matrix based pairwise comparisons of sodAint fragments of streptococcal type strains

Straina % Identity with the following strainb:
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1. S. acidominimus 71.3 67.8 71.7 68.0 69.9 69.9 70.8 73.3 67.6 65.3 66.0 69.9 72.6 70.8 68.5 69.7 72.6 70.8 66.9 69.0 70.3 68.0 72.0 69.7 70.1 79.5 73.3 73.3
2. S. agalactiae 69.0 72.0 72.6 71.7 73.1 71.3 71.5 71.5 66.7 66.7 74.0 73.3 71.7 71.0 69.7 74.5 69.9 72.4 70.1 71.3 70.8 74.5 73.1 74.0 74.7 72.4 72.6
3. S. alactolyticus 73.1 74.9 69.7 76.1 66.4 69.4 69.7 66.2 65.7 75.2 72.0 66.9 73.1 70.8 70.6 70.6 71.0 71.0 69.7 70.3 74.5 72.2 68.3 68.3 72.2 71.3
4. S. anginosus 71.7 67.1 86.4 69.4 68.5 65.5 66.9 66.7 72.2 80.5 65.3 83.4 75.9 69.7 74.3 75.9 75.2 66.9 66.7 69.2 76.6 66.7 72.4 70.8 70.3
5. S. bovis 72.0 70.3 72.9 70.6 68.7 66.4 66.4 97.9 70.6 68.0 69.9 72.9 74.0 71.7 73.3 72.2 68.5 70.6 77.5 69.4 69.7 70.3 75.9 76.3
6. S. canis 64.1 73.1 73.1 85.1 78.9 79.5 72.4 65.5 78.9 63.0 67.8 76.8 64.6 66.7 67.6 74.0 92.2 74.0 64.6 72.9 67.8 72.0 71.3
7. S. constellatus 65.5 66.4 66.0 62.3 62.1 71.5 81.8 66.9 90.1 76.8 68.7 75.6 78.2 76.1 69.2 66.7 70.3 79.8 65.3 71.5 71.5 70.6
8. S. cricetus 84.1 70.3 68.0 68.7 73.3 70.1 72.2 64.8 71.0 75.6 70.3 70.6 70.8 69.2 72.0 75.9 69.0 84.1 74.7 73.6 73.3
9. S. downei 70.1 71.0 70.8 70.1 65.7 73.1 63.9 68.3 74.0 66.9 66.2 68.7 69.7 71.3 73.8 68.7 85.5 76.8 73.1 73.1
10. S. dysgalactiae 74.9 74.9 69.0 62.8 77.5 64.6 66.9 69.9 66.4 67.6 67.1 72.4 86.9 72.0 65.3 73.1 65.7 73.1 71.5
11. S. equi subsp. equi 98.9 65.7 63.0 71.3 63.2 63.7 70.1 63.7 64.1 63.7 68.0 76.1 69.7 63.4 68.7 66.9 66.7 66.4
12. S. equi subsp. zooepidemicus 65.7 63.0 71.3 63.0 64.1 69.7 64.1 64.8 64.1 68.3 77.0 69.9 62.5 69.0 66.2 66.7 66.4
13. S. equinus 71.7 68.5 71.7 72.6 74.7 72.2 73.3 71.7 69.2 70.3 77.2 70.1 69.2 69.9 75.9 76.8
14. S. gordonii 64.1 81.1 80.5 70.3 79.8 80.9 80.2 66.7 63.9 73.3 85.7 68.7 72.9 73.3 73.1
15. S. iniae 63.9 66.0 74.0 66.4 65.7 66.9 77.0 79.3 72.4 65.3 70.3 71.0 70.8 70.1
16. S. intermedius 75.6 67.4 76.1 77.2 74.3 68.5 63.2 68.5 77.0 62.3 70.1 70.6 70.1
17. S. mitis 68.5 91.7 85.7 96.6 67.1 66.7 76.3 78.9 70.8 73.3 75.6 75.2
18. S. mutans 67.4 67.6 68.5 74.3 75.2 78.4 68.7 72.0 74.5 74.7 75.6
19. S. oralis 85.3 92.0 66.9 63.0 73.6 79.5 69.4 73.3 74.0 73.6
20. S. parasanguis 85.7 65.3 64.8 70.8 79.3 68.5 72.2 70.6 70.8
21. S. pneumoniae 66.4 66.7 75.4 78.2 71.3 73.6 75.2 74.9
22. S. porcinus 74.9 71.0 66.4 69.4 71.3 73.1 72.0
23. S. pyogenes 74.5 63.7 72.6 66.7 71.5 70.3
24. S. salivarius 72.2 75.6 73.3 89.7 90.1
25. S. sanguis 67.4 72.0 72.4 71.7
26. S. sobrinus 75.4 77.0 75.6
27. S. suis 72.0 72.9
28. S. thermophilus 96.8
29. S. vestibularis
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a

The main characteristics of the strains are listed in Table 1. 

b

The strain numbers correspond to the strains identified by the numbers on the left. 

Species identification of streptococcal clinical isolates by sequencing the sodAint 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 sodAint 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 sodAint fragment on the phylogenetic tree of the sodAint 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 sodAint systems (Table 2). We also include in this study the sequences of sodAint fragments of known streptococcal species present in the databases. The results obtained with the sodAint 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 sodAint-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 sodAint system as S. anginosus and S. oralis, respectively (Table 2). The sequence of the sodAint 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 sodAint 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 sodAint-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 sodAint system but not with ID 32 Strep system (Table 2). Finally, it is important that the intraspecies divergence between the sodAint 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 sodAint fragments can exceed those observed between the sodAint 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 sodAint 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 sodAint 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 sodAint 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 sodAint fragments from other species of streptococci as well as from other related genera such as Abiotrophia, Enterococcus, Gemella, Leuconostoc, and Pediococcus.

FIG. 3.

FIG. 3

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Electropherograms showing part of the nucleotide sequence of sodAint from S. porcinus. Sequence reactions were carried out with a pUC18ΩsodAint 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.

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