Identification of Clinically Relevant Viridans Group Streptococci by Sequence Analysis of the 16S 23S Ribosomal DNA Spacer Region

Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Clinically Relevant Viridans Group Streptococci by

Sequence Analysis of the 16S-23S Ribosomal DNA Spacer Region

Chao Chien Chen,

_{Lee Jene Teng,}

_{and Tsung Chain Chang}

_*

Medical Technology, National Taiwan University College of Medicine, Taipei,2_{and Department of Medical Technology,} School of Medicine, National Cheng Kung University, Tainan,3_{Taiwan, Republic of China}

Received 15 August 2003/Returned for modification 15 December 2003/Accepted 2 March 2004

The feasibility of sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (ITS) for the identification of clinically relevant viridans group streptococci (VS) was evaluated. The ITS regions of 29

reference strains (11 species) of VS were amplified by PCR and sequenced. These 11 species wereStreptococcus

anginosus,S. constellatus,S. gordonii,S. intermedius,S. mitis,S. mutans,S. oralis,S. parasanguinis,S. salivarius,

S. sanguinis, andS. uberis. The ITS lengths (246 to 391 bp) and sequences were highly conserved among strains within a species. The intraspecies similarity scores for the ITS sequences ranged from 0.98 to 1.0, except for

the score forS. gordoniistrains. The interspecies similarity scores for the ITS sequences varied from 0.31 to

0.93. Phylogenetic analysis of the ITS regions revealed that evolution of the regions of some species of VS is not parallel to that of the 16S rRNA genes. One hundred six clinical isolates of VS were identified by the Rapid ID 32 STREP system (bioMe´rieux Vitek, Marcy l’Etoile, France) and by ITS sequencing, and the level of disagreement between the two methods was 18% (19 isolates). Most isolates producing discrepant results could be unambiguously assigned to a specific species by their ITS sequences. The accuracy of using ITS sequencing

for identification of VS was verified by 16S rDNA sequencing for all strains except strains ofS. oralisandS.

mitis, which were difficult to differentiate by their 16S rDNA sequences. In conclusion, identification of species of VS by ITS sequencing is reliable and could be used as an alternative accurate method for identification of VS.

Viridans group streptococci (VS) are the most common eti-ologic agents of subacute infective endocarditis and are capa-ble of causing a variety of pyogenic infections (9). VS can be divided into five major groups on the basis of the 16S ribo-somal DNA (rDNA) sequences: (i) the mutans group, (ii) the salivarius group, (iii) the anginosus group (also called the mil-leri group), (iv) the sanguinis group, and (v) the mitis group (9).

VS can be isolated as part of the normal flora of the respi-ratory, genital, and alimentary tracts. However, species of VS are playing an increasing role in infections among immuno-compromised patients (33). The clinical significance of VS may differ between species, and sometimes it is important to iden-tify the individual species associated with diseases (18). Jacobs et al. (19) studied 104 isolates of VS recovered from blood

cultures and found thatStreptococcus oralisand S. mitiswere

the two species most frequently isolated from patients in the hematology unit, whereas species of the anginosus group were the most common species isolated from the general hospital population. Chang et al. (7) found that VS accounted for 9% of all cases of culture-proven bacterial meningitis in adults, and more than 80% of VS causing meningitis were species of the anginosus group. These data provided further insight into the search for the link between species and the diseases that they can cause.

The antimicrobial susceptibilities of VS isolated from

pa-tients with significant infections in Taiwan revealed that

high-level penicillin resistance (MICs ⱖ 4 ␮g/ml) was frequently

found in isolates ofS. oralis(35%) andS. mitis(20%).

Mac-rolide resistance also occurred most frequently in S. oralis

isolates (55%) but in none ofS. mutansisolates (35). However,

Tracy et al. (38) found that there were no differences in

anti-microbial susceptibilities among species (S. anginosus,S.

con-stellatus, and S. intermedius) of the anginosus group. Those investigators expressed the opinion that it was unnecessary to identify VS to the species level and that identification of an infecting organism as a member of the anginosus group might be sufficient for patient management, but whether the same can be said for other VS remains to be established (38).

Nearly 40 conventional tests have been used to differentiate species of VS, but phenotypic tests do not allow an unequivocal identification of some species of this heterogeneous group of bacteria (1, 10, 23). This is because variability in a common phenotypic trait among strains is common (4, 17, 22). Species of S. mutans strains and strains of the anginosus and mitis groups are the most problematic to differentiate (11, 18, 32).

Several molecular methods have been developed for the identification of VS to the species level (5, 11, 12, 18, 21, 29, 36). The targets include the rRNA genes (6, 8, 18, 20, 30), the tRNA gene intergenic spacer (ITS) (12), and the genes

encod-ingD-alanine–D-alanine ligase (11), manganese-dependent

su-peroxide dismutase (sodAint) (26), and heat shock proteins

(groESL) (34). A variety of techniques, including DNA hybrid-ization (18, 21, 30, 32), PCR product length polymorphism analysis (12), arbitrarily primed PCR (29), and comparative DNA sequence analysis, have been used for molecular diag-nosis (6, 8, 26, 36). Compared with the lengths of 16S rDNA

(⬇1.5 kb) (27) andgroESL(⬇2 kb) (36), the ITS lengths of VS

* Corresponding author. Mailing address: Department of Medical Technology, School of Medicine, National Cheng Kung University, 1 University Rd., Tainan 701, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5790. Fax: 886-6-2363956. E-mail: [email protected] .edu.tw.

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are relatively short (⬍400 bp). In addition,S. mitisandS. oralis

cannot be differentiated by analyzing the groESL (36) and

sodAintgenes (26), since intraspecies sequence variations are

higher than interspecies variations.

The ITS separating the 16S and 23S rDNAs has been sug-gested to be a good candidate for use in molecular assays for bacterial species identification (3, 39) and strain typing (15). Sequences of the ITS regions have been found to have low levels of intraspecies variation and high levels of interspecies divergence (40). The aim of this study was to investigate the feasibility of using the ITS sequences to identify 11 clinically relevant species of VS.

MATERIALS AND METHODS

Bacterial strains. Twenty-nine type or reference strains of VS, includingS. anginosus,S. constellatus,S. gordonii,S. intermedius,S. mitis,S. mutans,S. oralis,

S. parasanguinis,S. salivarius,S. sanguinis, andS. uberis, were used in this study (Table 1). Most of these strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.).S. intermediusNCDO 2227 was obtained from the National Collection of Food Bacteria, Reading, United Kingdom. In addition, type strains ofS. agalactiae(ATCC 13813),S. pneumoniae(ATCC 33400),S. pyogenes(ATCC 14289), andEnterococcus faecalis(ATCC 19433) and two reference strains ofS. pneumoniae(ATCC 27336 and ATCC 49169) were also used for comparison of their ITS sequences with those of VS. A total of 106 clinical isolates of VS were obtained from National Taiwan University Hospital (Taipei, Taiwan) and National Cheng Kung University Medical Center (Tainan, Taiwan). Most of the clinical isolates were recovered from blood cultures and deep abscesses (e.g., brain abscesses) and were phenotypically identified with the Rapid ID 32 STREP system (bioMe´rieux Vitek, Marcy l’Etoile, France).

DNA preparation.The boiling method described by Millar et al. (25) was used to extract DNA from the bacteria. Briefly, one colony from pure cultures was suspended in 20␮l of sterilized water and heated at 100°C for 15 min in a heating block. After centrifugation in a microcentrifuge (6,000⫻g for 3 min), the supernatant containing bacterial DNA was stored at⫺20°C for further use.

Amplification of ITS regions and nucleotide sequence determination.The bacterium-specific universal primers 13BF (5⬘-GTGAA TACGT TCCCG GGC CT-3⬘) (27) and 6R (5⬘-GGGTT YCCCC RTTCR GAAAT-3⬘) (15) (where Y is C or T and R is A or G) were used to amplify a DNA fragment that encompassed a small portion of the 16S rDNA region, the ITS, and a small portion of the 23S rDNA region. The 5⬘end of primer 13BF is located at position 1371 of the 16S rDNA, and the 5⬘end of primer 6R is located at position 108 downstream of the 5⬘end of the 23S rDNA (Escherichia colinumbering). PCR was performed with 5␮l (1 to 5 ng) of template DNA in a total reaction volume of 50␮l consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 0.1␮M (each) primer, 1 U ofTaqDNA polymerase, and 50␮l of a mineral oil overlay. The PCR program consisted of 8 cycles of denaturation (94°C for 2 min), annealing (55°C for 1 min), and extension (72°C for 1 min), followed by 30 cycles of denaturation (94°C for 1 min), annealing (60°C for 1 min), and extension (72°C for 1 min) and a final extension step at 72°C for 3 min. An OmniGen thermal cycler (Hybaid Limited, Winsford, United Kingdom) was used for PCR.

The PCR products were purified with a PCR-M Clean Up kit (Viogene, Taipei, Taiwan) and were sequenced on a model 377 sequencing system (Applied Biosystems, Taipei, Taiwan) with a BigDye Terminator Cycle Sequencing kit (version 3.1; Applied Biosystems). After sequencing of the PCR products, por-tions of the 16S and 23S rDNA regions were removed to obtain the full ITS sequences. For all VS studied, the 5⬘ends of the ITS sequences were CTAAGG, whereas the 3⬘ends of the ITS sequences were AATAA. The similarity score for the ITS sequence of a strain was obtained by comparing its sequence with that of the type strain of the same species, and the PileUp command of the Wisconsin Genetics Computer Group (GCG) package (version 10.3; Accelrys Inc., San Diego, Calif.) was used for calculation. The PrettyBox program of the GCG package was used to align multiple ITS sequences.

Phylogenetic analysis.Multiple-sequence alignment of the ITS sequences was carried out with the CLUSTAL X program (version 1.63b) (37), and an unrooted phylogenetic tree was constructed by using the neighbor-joining method (31) listed in the MEGA (molecular evolutionary genetic analysis; version 2.1) ana-lytical software package (24). For neighbor-joining analysis, the distance between the sequences was calculated by using Kimura’s two-parameter model. The

TABLE 1. Intraspecies similarity scores for the 16S-23S rDNA ITSs of different species of VS

Species Strain no.a Size of

ITS (bp) Similarityscoreb _{accession no.}GenBank c

S. anginosus ATCC 33397d _{391 AY347541}

ATCC 700231 390 0.99 AY351328 NCTC 10713e _{391 0.99 L36931}

S. constellatus ATCC 27513d _{391 AY347545}

ATCC 27823 390 0.99 AY347546 NCDO 2226e _{391 0.99 L36933}

S. gordonii ATCC 10558d _{246 AY353081}

ATCC 10558e _{246 1.00 AB051019} ATCC 49818 248 0.94 AY351328 ATCC 35105 248 0.94 AY351326

S. intermedius ATCC 27335d _{370 AY347549}

NCDO 2227 370 1.00 L36934

S. mitis ATCC 49456d _{248 AY347550}

SK24e _{249 0.99 AB051020}

S. mutans ATCC 31377d _{388 AY351323}

ATCC 31383 387 0.98 AY351324

GS5f _{387 0.99 AY351312}

MT4251e _{388 0.99 AB050990}

MT4245e _{388 0.99 AB050988}

MT8148e _{388 0.99 AB050989}

S. oralis ATCC 35037d _{246 AY347551}

ATCC 55229 246 0.99 AY347553 ATCC 700233 246 0.99 AY347554 ATCC 700234 246 0.99 AY347555

ATCC 9811 246 0.99 AY347568

ATCC 10557e _{246 0.99 AB051018}

S. parasanguinis ATCC 15912d _{249 AY351320}

ATCC 15909 249 0.99 AY351319

S. salivarius ATCC 13419d _{273 AY347562}

ATCC 7073 273 0.99 AY347561

ATCC 25975 273 1.00 AY347564 ATCC 15910 273 0.99 AY347563 DSM 20560e _{273 0.99 AJ439458} NCTC 8618e _{273 0.98 AB051016}

S. sanguinis ATCC 10556d _{264 AY347565}

UASsB22e _{264 1.00 AF271353}

UASsB25e _{264 1.00 AF271355}

UASsB29e _{264 0.99 AF271351}

UASsB30e _{264 1.00 AF271354}

UASsB47e _{264 0.99 AF271352}

S. uberis ATCC 13386d _{340 AY347566}

ATCC 700407 338 0.97 AY347567 ATCC 19436 340 0.99 AY347538 NCDO 2038e _{338 0.99 AF255657} ATCC 27958e _{340 0.99 U39768}

a_{DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig, Germany;}

NCDO, National Collection of Food Bacteria, Reading, United Kingdom; NCTC, National Collection of Type Cultures, Public Health Laboratory, Lon-don, United Kingdom.

b_{The type strains were used as the basis for calculation of similarity scores.} c_{The ITS sequences (GenBank accession numbers) ofS. agalactiaeATCC}

13813 (AY347539), S. pneumoniaeATCC 33400 (AY347557), ATCC 27336 (AY347556), ATCC 49169 (AY347558),S. pyogenesATCC 14289 (AY347560), andE. faecalisATCC 19433 (AY351321) were also submitted to GenBank.

d_{Type strain.}

e_{Strains for which ITS sequences are available in the GenBank database.} f_{Clinical isolate.}

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robustness of the neighbor-joining method was statistically evaluated by boot-strap analysis with 1,000 bootboot-strap samples.

Identification of clinical isolates of VS by ITS sequencing.A total of 106 clinical isolates were tested to evaluate the feasibility of using ITS sequencing for VS identification. These isolates were first identified with the Rapid ID 32 STREP system (bioMe´rieux Vitek). The ITS sequence of a clinical isolate was compared to those of the type strains (Table 1), and a species name was given according to the highest similarity score obtained. Clinical isolates for which the Rapid ID 32 STREP system and ITS sequence analysis produced discrepant identifications were further identified according to their 16S rDNA sequences.

Species identification by 16S rDNA sequencing.Primer pair 8FPL (5⬘-GTTT GATCCTGGCTCAG-3⬘) and 1492RPL (5⬘-GGTTACCTTGTTACGACTT-3⬘) was used for PCR amplification (27) of the nearly complete length of the 16S rDNA of clinical isolates for which the Rapid ID 32 STREP system and ITS sequencing produced discrepant identifications. The amplicons were cycle se-quenced in both directions by using the two primers described above and an additional primer, primer 1055r (5⬘-CACGAGCTGACGACAGCCAT-3⬘). The sequences determined were compared to known 16S rRNA gene sequences in the National Center for Biotechnology Information database by using the BLAST algorithm.

Nucleotide sequence accession numbers.The ITS sequences of 29 reference strains and 1 clinical isolate (a total of 30 sequences) of VS and several strep-tococcal species were submitted to GenBank. The accession numbers are listed in Table 1.

RESULTS

Amplification and sequencing of the ITS fragments. The ITS fragments of 11 species (29 reference strains) of clinically rel-evant VS were amplified by PCR with primers 13BF and 6R. A single amplicon was observed for each species (Fig. 1). After sequencing and removal of the partial 16S and 23S rDNA portions, the exact lengths and sequences of the ITSs were

obtained. S. oralis had the shortest ITS fragment (246 bp),

followed byS. mitis(248 to 249 bp), whereasS. constellatusand

S. anginosusyielded the longest ITS fragments (390 to 391 bp). Intraspecies variations in ITS lengths were within 3 bp (Table 1).

Sequence similarities of ITS regions. Generally, the

in-traspecies similarity scores were very high, ranging from 0.97 to

1.0 (Table 1). However, an exception was found forS. gordonii;

two of the three reference strains (strains ATCC 49818 and ATCC 35105) had similarity scores of only 0.94 each when their sequences were compared with that of the type strain (ATCC 10558). In addition to the 11 species of VS, the ITS accession numbers of several commonly encountered

strepto-coccal species and E. faecalisare also listed in footnotec of

Table 1. Some ITS sequences of VS available in GenBank are also included in Table 1 to facilitate intraspecies comparisons. Table 1 demonstrates the high degree of intraspecies conser-vation of both the lengths and the sequences of the ITS re-gions.

Table 2 shows the results of pairwise comparisons of the ITS sequences between type strains of any two given species. In

addition to the 11 species of VS, type strains ofS. agalactiae,S.

pyogenes,S. pneumoniae, andE. faecaliswere also included for comparison. Table 2 reveals a high level of interspecies diver-gence of the ITS sequences. Among the type strains of VS, the

similarity scores ranged from 0.51 (S. mutans versusS.

angino-sus) to 0.93 (S. mitisversusS. oralis). However, a high similarity

score (0.94) between type strains ofS. pneumoniaeandS. mitis

was also observed (Table 2). In general, the interspecies ITS similarity scores were less than 0.80.

Table 3 shows the results of intra- and interspecies

compar-FIG. 1. Amplification of VS type strains with primers 13BF and 6R and separation of the PCR products by 2% agarose gel electrophore-sis. Lanes: 1,S. mitis; 2,S. oralis; 3,S. constellatus; 4,S. intermedius; 5,

S. salivarius; 6,S. anginosus; 7,S. mutans; 8,S. sanguinis; 9,S. gordonii; 10,S. parasanguis; 11,S. uberis; M, 100-bp ladder.

TABLE 2. ITS sequence similarity scores among type strains of streptococcal and enterococcal species

Species no. and

species ATCCno.

Sequence similarity score for the following speciesa_:

2 3 4 5 6 7 8 9 10 11 12 13 14 15

1.S. agalactiae 13813 0.58 0.57 0.68 0.62 0.65 0.56 0.64 0.66 0.64 0.55 0.76 0.56 0.52 0.35

2.S. anginosus 33397 0.53 0.62 0.61 0.60 0.51 0.61 0.61 0.56 0.74 0.57 0.54 0.48 0.35

3.S. constellatus 27513 0.61 0.66 0.58 0.53 0.59 0.63 0.56 0.53 0.57 0.55 0.51 0.36

4.S. gordonii 10558 0.71 0.73 0.60 0.76 0.76 0.72 0.58 0.70 0.76 0.54 0.32

5.S. intermedius 27335 0.69 0.56 0.69 0.69 0.66 0.57 0.62 0.70 0.53 0.34

6.S. mitis 49456 0.58 0.93 0.75 0.94 0.61 0.67 0.72 0.56 0.36

7.S. mutans 31377 0.58 0.58 0.55 0.53 0.57 0.53 0.46 0.35

8.S. oralis 35037 0.77 0.90 0.60 0.68 0.73 0.56 0.35

9.S. parasanguinis 15912 0.72 0.60 0.68 0.73 0.51 0.35

10.S. pneumoniae 33400 0.57 0.67 0.72 0.52 0.35

11.S. pyogenes 14289 0.55 0.53 0.49 0.33

12.S. salivarius 13419 0.54 0.52 0.36

13.S. sanguinis 10556 0.51 0.37

14.S. uberis 13386 0.34

15.E. faecalis 19433

a_{The species numbers correspond to the species numbers identified on the left.}

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isons of the ITS sequences between strains ofS. mitis,S. oralis, andS. pneumoniae. It was noted thatS. pneumoniaecould not

be differentiated fromS. mitisby the similarity scores for the

ITS sequences. For example, the similarity score between the

type strains ofS. pneumoniaeATCC 33400 andS. mitisATCC

49456 (0.99) was higher than the intraspecies similarity score

betweenS. mitisATCC 49456 andS. mitisSK24 (0.98).

The ITS sequences ofS. mitisand S. oralisalso have high

similarity scores. The intraspecies similarity scores for the ITS

regions ranged from 0.98 to 1.0 and from 0.99 to 1.0 forS. mitis

and S. oralisstrains, respectively (Table 3). However, the in-terspecies similarity scores between strains of the two species (0.93 to 0.95) were consistently lower than those observed for

strains within each species. Therefore,S. mitisstrains could be

effectively differentiated from S. oralisstrains on the basis of

their ITS sequences. Moreover, it was interesting that all six

reference strains ofS. oralishad a single deletion at ITS

nu-cleotide positions 199 and 217 ofS. mitis; however, the

nucle-otides were A and T at these two positions in the type strain of

S. mitis(ATCC 49456) andS. mitisSK24 (GenBank accession no. AB051020), respectively. Another important characteristic

that could be used to differentiateS. mitisfromS. oraliswas

that the ITS lengths inS. mitis(248 to 249 bp) were consistently

2 to 3 bp longer than those inS. oralis(246 bp) (Table 1).

Phylogenetic relationships. The phylogenetic relationships

derived from the ITS sequences of the 11 type strains of VS

and the type strains of S. pneumoniae and S. pyogenes are

presented in Fig. 2. The phylogenetic tree constructed revealed

that strains of the mitis group (S. mitis and S. oralis) and S.

pneumoniaewere highly related and grouped together.

How-ever, it was noted thatS. parasanguinisdid not cluster with the

other two species (S. gordoniiandS. sanguinis) constituting the

sanguinis group. In addition,S. anginosuswas genetically

sep-arated fromS. constellatusandS. intermediusof the anginosus

TABLE 3. Intraspecies and interspecies ITS sequence similarity scores among strains ofS. mitis, S. oralis,andS. pneumoniae

Species no. and

species ATCCno.

Sequence similarity score for the following speciesa_:

2 3 4 5 6 7 8 9 10 11 12 13

1.S. mitis 49456b _{0.98 0.94 0.95 0.94 0.95 0.94 0.94 0.95 0.95 0.99 0.95 1.00}

2.S. mitis SK24c _{0.93 0.93 0.93 0.93 0.93 0.93 0.93 0.94 0.99 0.94 0.99}

3.S. oralis 49296 1.00 1.00 1.00 0.99 0.99 1.00 0.90 0.93 0.90 0.95

4.S. oralis 9811 1.00 1.00 0.99 0.99 1.00 0.89 0.93 0.89 0.94

5.S. oralis 10557c _{1.00 0.99 0.99 1.00 0.90 0.93 0.90 0.95}

6.S. oralis 35037b _{0.99 0.99 1.00 0.89 0.93 0.89 0.94}

7.S. oralis 55229 1.00 0.99 0.90 0.93 0.90 0.95

8.S. oralis 700233 0.99 0.90 0.93 0.90 0.95

9.S. oralis 700234 0.89 0.93 0.89 0.94

10.S. pneumoniae M60763c _{0.93 0.99 0.95}

11.S. pneumoniae 33400b _{0.93 0.98}

12.S. pneumoniae 27336 0.95

13.S. pneumoniae 49169

a_{The species numbers correspond to the species numbers identified on the left.} b_{Type strains.}

c_{Strains that were not obtained from ATCC but whose ITS sequences were available in the GenBank database.}

FIG. 2. Phylogenetic relationship of various species of VS,S. pneumoniae, andS. pyogeneson the basis of the ITS sequences. The phylogenetic tree was generated by the neighbor-joining method within the MEGA package. The numbers at the nodes are the percentages of occurrence in 1,000 bootstrapped resamplings.

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group and from the other nine species of VS in the phyloge-netic tree.

Identification of clinical isolates of VS by ITS sequencing.A

total of 106 clinical isolates were analyzed to validate ITS sequencing for identification of VS. Overall, the species iden-tities obtained with the Rapid ID 32 STREP system and by ITS sequence analysis showed 82% (87 isolates) agreement. Con-cordant identifications were obtained for the following species

(with the numbers of isolates indicated in parentheses): S.

anginosus(n⫽11),S. constellatus(n⫽12),S. gordonii(n⫽2),

S. intermedius(n⫽12),S. mitis(n⫽9),S. mutans(n⫽8),S. oralis(n⫽11),S. parasanguinis(n⫽1),S. salivarius(n⫽11),

S. sanguinis(n⫽8), andS. uberis(n⫽2). It was interesting

that all clinical isolates ofS. mitisandS. oralisanalyzed in this

study possessed the same characteristics as the reference strains, i.e., either the presence or the absence of A and T residues at ITS nucleotide positions 199 and 217, respectively. The 19 (18%) clinical isolates for which the Rapid ID 32 STREP system and ITS sequencing produced discrepant iden-tifications are listed in Table 4. Strains that produced discrep-ant results were usually given an “unacceptable profile” or low identification percentages with the Rapid ID 32 STREP sys-tem. Conversely, the ITS sequence identities were greater than 96.7% for all strains with discrepant results (Table 4), as com-parisons were made between these strains and the respective type strains. The strains for which discrepant identifications

tended to be produced were species of the anginosus group (S.

anginosus, S. constellatus, andS. intermedius), species of the

mitis group (S. mitisandS. oralis), andS. gordonii(Table 4).

Nine discrepant results were caused by the identification ofS.

parasanguinis strains with the Rapid ID 32 STREP system.

Four strains phenotypically identified asS. intermediuswith the

Rapid ID 32 STREP system were determined to beS.

angino-susby the present molecular approach.

The 19 strains with discrepant results were further identified

by 16S rDNA sequencing. Except for strains ofS. mitisandS.

oralis, the identities of all 11 strains of other species identified by ITS sequencing were confirmed by sequencing of their 16S

rDNA (Table 4). Among the seven strains identified asS. mitis

or S. oralis by ITS sequencing, their 16S rDNA sequences

always matched those of bothS. mitisandS. oralis with very

high scores by use of database searches with the BLAST pro-gram. The ITS sequence of strain 1590 had a similarity of 99%

with that ofS. gallactolyticusATCC 9809, whose ITS sequence

we sequenced and submitted to GenBank (GenBank accession no. AY347542). The 16S rDNA sequence of strain 1590 also

had 99% identity with that ofS. gallactolyticusATCC 43143 in

the GenBank database. Strain 7520 was not identified by ITS

sequencing; however, it was identified as S. sanguinis and S.

siemenswith the Rapid ID 32 STREP system and by 16S rDNA sequencing, respectively. Because the 16S rDNA sequence of

strain 7520 had a 99% identity with that ofS. siemens(strain

HK; GenBank accession no. AF432856), this strain was

con-sidered to beS. siemens.

DISCUSSION

This study has described a method that could identify clin-ically relevant VS to the species level. The method consists of (i) a PCR with two universal primers for amplification of a fragment encompassing the ITS sequence and partial regions of the 16S and 23S rDNAs, (ii) sequencing of the amplicon, and (iii) comparison of the ITS sequence with those of the type and reference strains of VS. The whole procedure can be

TABLE 4. Clinical isolates of VS for which discrepant identifications were produced with the Rapid ID 32 STREP system and by ITS sequence analysis

Strain

Species identification by:

ITS sequencinga Rapid ID 32 STREP

systemb 16S rDNA sequencingc

1590 S. gallolyticus(99) NId_{(UA) S. gallolyticus(99)}

336 S. anginosus(99.5) S. parasanguinis(UA) S. anginosus(99)

5437 S. anginosus(96.7) S. intermedius(98.2) S. anginosus(99.3)

6347 S. anginosus(98.4) S. intermedius(97.3) S. anginosus(96)

7940 S. anginosus(98.4) S. oralis(UA) S. anginosus(99)

8234-2 S. anginosus(99.5) S. intermedius(98.7) S. anginosus(99)

MyP700 S. anginosus(96.7) S. intermedius(98.2) S. anginosus(98)

737 S. constellatus(100) S. parasanguinis(UA) S. constellatus(98)

4938 S. constellatus(99.6) S. parasanguinis(UA) S. constellatus(99)

4672 S. gordonii(98.7) S. parasanguinis(UA) S. gordonii(99)

6826 S. gordonii(99.1) S. parasanguinis(UA) S. gordonii(97.7)

2000 S. mitis(99.1) S. parasanguinis(UA) S. oralis(98),S. mitis(98)

8916 S. mitis(99.1) S. oralis(UA) S. mitis(99),S. sanguinis(99)

3788-2 S. mitis(99.1) S. parasanguinis(UA) S. mitis(98),S. oralis(97)

8903-2 S. mitis(98.3) S. thermophilus(UA) S. mitis(98),S. pneumoniae(98)

8984 S. oralis(100) S. mitis(97.5) S. mitis(97),S. oralis(97)

5881-2 S. oralis(99.5) S. parasanguinis(72) S. mitis(99.8),S. oralis(98.4)

5129-2 S. oralis(99.5) S. parasanguinis(91) S. mitis(98),S. oralis(97.7)

7520 NI S. sanguinis(89.1) S. siemens(99)

a_{The values in parentheses are the percent identities of the strains studied compared to the sequence of the corresponding type strain.} b_{The values in parentheses are the percent identities obtained with the Rapid ID 32 STREP system. UA, unacceptable profile.} c_{The values in parentheses are the percent 16S rDNA sequence identities with the sequences available in GenBank.} d_{NI, not identified.}

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finished within 24 h if isolated colonies are available. This method might be useful under conditions that necessitate iden-tification of VS to the species level. It provides an accurate alternative for the delineation of some species of VS that are difficult to identify.

PCR amplification of the ITS regions depends on the occur-rence of highly conserved regions in the flanking termini of the 16S and 23S rRNA genes. The primers used in this study (primers 13BF and 6R) also could efficiently amplify the ITS regions of nutritionally variant streptococci and streptococci other than VS by using the PCR conditions specified here (data not shown). Whiley et al. (40) found that some atypical strains of the anginosus group produced PCR products whose lengths were different from those of “normal” strains. How-ever, most of these atypical strains were eventually found to be misclassified, as further examined by DNA-DNA reassociation experiments (40).

Pairwise comparison of two given species of VS revealed a lower level of sequence similarity between their ITS regions (Table 2) than between their 16S rDNA sequences (20). These results indicate that the ITS region might constitute a more discriminative target sequence than 16S rDNA for the differ-entiation of closely related species of VS. It was found that considerable variation in both the lengths and the sequences of the ITS regions can occur between species (14). The ITS re-gion has been suggested to be a suitable target from which useful taxonomic information for bacteria can be derived, par-ticularly with regard to identification to the species level (3, 16, 28, 34, 39) and genotyping (13, 14). A limitation for bacterial identification based on ITS sequencing is the limited availabil-ity of ITS sequences; however, the numbers of ITS region sequences available in public databases has increased rapidly in recent years.

16S rDNA sequencing (sequence length, approximately 1.5 kb) is well established as a standard method for the identifi-cation of species, genera, and families of bacteria (2). How-ever, the ITS lengths of VS are relatively short (246 to 391 bp) (Table 1), and sequencing of the ITS region would be easier and more straightforward. In this study, each of the 11 clini-cally important species of VS produced a single PCR product that facilitated direct sequencing (Fig. 1).

In this study, four strains identified asS. intermediuswith the

Rapid ID 32 STREP system were actually S. anginosus, as

revealed by their ITS and 16S rDNA sequences. However,

strains identified as S. parasanguinis with the Rapid ID 32

STREP system could be S. anginosus, S. constellatus, or S.

gordonii(Table 4). Jacobs et al. (19) reported that bloodstream

isolates ofS. anginosusare frequently identified asS.

interme-diuswith the Rapid ID 32 STREP system.

ThegroESL(36) andsodAint(26) genes have been used for differentiation of species of VS and streptococci, respectively. Since sequence heterogeneity of these two genes exists among different species, highly degenerate primers were used to am-plify the two genes from different species. The disadvantage of using highly degenerate primers is that the annealing temper-ature of the PCR must be low enough to ensure effective pairing of the primers and the template DNA. However, under low-stringency conditions, the chances of producing aberrant

PCR products might increase. The drawback of using the

so-dAint gene for the identification of streptococci is that the

intraspecies sequence divergence may vary greatly, depending upon the species considered. Therefore, the identification of species can be made only by determining the positions of their

sodAintsequences on the phylogenetic tree constructed from

type strains (26). In addition,S. mitisand S. oraliscannot be

differentiated by sequence analysis of the groESL (36) and

sodAint(26) genes, since intraspecies sequence variations are

greater than interspecies variations.

Kikuchi et al. (21) found that among the 17 strains identified asS. mitiswith the Rapid ID 32 STREP system, the results for only 3 strains were in agreement with the results of DNA-DNA hybridization. Jacobs et al. (19) reported that bloodstream

isolates identified asS. oralisare mainly those previously

iden-tified as S. mitis. The 16S rDNA sequence identities of type

strains ofS. oralis(ATCC 35037) andS. mitis(ATCC 49456)

are greater than 99% (20). After pairwise comparisons of the

16S rDNA sequences of six reference strains ofS. mitis(ATCC

903, ATCC 6249, ATCC 15914, ATCC 49456, NCTC 12261,

and NCTC 3165) and four reference strains ofS. oralis(ATCC

700233, ATCC 9811, ATCC 35037, and NCTC 11427) avail-able in the GenBank database, it was found that the intraspe-cies sequence variations (0.93 to 1.0) were higher than the interspecies sequence variations (0.96 to 1.0). Therefore,

iden-tification of S. mitis and S. oralison the basis of 16S rDNA

sequence analysis may not be reliable. This phenomenon was clearly demonstrated in the present study (Table 4). An anal-ysis with the BLAST algorithm showed that the 16S rDNA

sequences of strains identified as S. mitis by their ITS

se-quences always matched the 16S rRNA gene sese-quences of both

S. mitisandS. oraliswith high degrees of identity. This was also

true forS. oralisstrains.

However,S. mitiscould easily be differentiated fromS. oralis

by ITS sequence comparison (Table 3) or sequence alignment. The intraspecies ITS similarity scores for both species were higher than the interspecies ITS similarity scores (Table 3). In

addition, all seven reference strains ofS. oralishad one

dele-tion at ITS nucleotide posidele-tions 199 and 217, and this charac-teristic could be used as a fingerprint to differentiate species of

S. oralisandS. mitis. However, the intraspecies ITS divergence ofS. mitisandS. pneumoniaewas higher than the interspecies divergence of the two species (Table 3). Therefore, simple biochemical tests such as optochin susceptibility and bile

sol-ubility tests should be used to differentiateS. pneumoniaefrom

S. mitis; both tests are positive forS. pneumoniae, whereasS. mitisis optochin resistant and bile insoluble (9).

From the phylogenetic tree constructed by use of the ITS

sequences, S. parasanguinis and S. anginosusdid not cluster

with their respective groups (the sanguinis group and the

an-ginosus group, respectively). However,S. mitis,S. oralis, andS.

pneumoniaewere related and grouped together (Fig. 2). These three species were also grouped together when the genes

en-codinggroESL(36),sodAint(26), and 16S rRNA (6, 20) were

analyzed. Many bootstrap values shown in the phylogenetic tree (Fig. 2) were less than 70%; therefore, the ITS region may not be a good target for constructing a phylogenetic tree for species of VS. This implies that there exists an unparallel evolution between the ITS region and the 16S rRNA gene that is essential for the survival of all microorganisms.

In conclusion, identification of species of VS by ITS

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quencing seems to be reliable and could be used as an accurate alternative for the identification of VS.

ACKNOWLEDGMENTS

This project was supported by a grant (grant NSC 88-2314-B006-078) from the National Science Council and in part by a grant from the Department of Medical Research of the National Taiwan University Hospital, Taipei, Taiwan.

We thank S. Liu for help with constructing the phylogenetic tree and S. K. Tung for assistance with 16S rDNA sequencing.

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