JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schlegel, L.
Right arrow Articles by Bouvet, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schlegel, L.
Right arrow Articles by Bouvet, A.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2003, p. 657-666, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.657-666.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of Major Streptococcal Species by rrn-Amplified Ribosomal DNA Restriction Analysis

Laurent Schlegel,1,2 Francine Grimont,2 Patrick A. D. Grimont,2 and Anne Bouvet1*

Centre National de Référence des Streptocoques, Service de Microbiologie, Hôtel Dieu, AP-HP Université Paris VI,1 Unité Biodiversité des Bactéries Pathogènes Emergentes, INSERM U389, Institut Pasteur, Paris, France2

Received 20 May 2002/ Returned for modification 6 August 2002/ Accepted 31 October 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Amplified ribosomal DNA restriction analysis (rrn-ARDRA) is based on PCR amplification and restriction of a fragment of rRNA genes including 16S and 23S genes and the intergenic spacer. rrn-ARDRA was evaluated for the identification of species within the genus Streptococcus. A total of 148 type and reference strains of pyogenic, oral, and group D streptococci were examined in order to construct a database for identification of streptococci. The amplified product was a single band approximately 4,500 bp long. This amplicon was digested separately with three (HhaI, MboII, and Sau3A) restriction endonucleases. Respectively, 27, 26, and 28 major patterns were observed after HhaI, MboII, and Sau3A restrictions. Streptococcal strains belonging to different species had different patterns or different combination of patterns. An identification system based upon a combination of the three restriction patterns in a single database was then proposed. rrn-ARDRA was successfully applied to 11 clinical isolates whose identification to the species level was difficult to obtain by phenotypic analysis. Using a database of well-characterized strains, rrn-ARDRA is a powerful method for the identification of streptococcal isolates.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Streptococci are major pathogens for animals and human beings. Phenotypic studies and DNA-DNA hybridization assays have demonstrated that the genus Streptococcus may be divided in three major groups of species: pyogenic, oral, and group D streptococci (5, 6, 17, 20, 21, 27).

Identification of streptococcal species is currently based on observation of the cultural and morphological characteristics, determination of the biochemical pattern (production of enzymes and production of acid from various carbohydrate sources) (4, 5, 6, 9, 11, 17, 20), and observation of the antigenic structure according to the classification of Rebecca Lancefield (22).

Molecular tools as ribotyping (3, 8, 25, 26), oligonucleotide probing (2), PCR-based protocols (13, 23), and DNA sequence analysis (1, 19, 24) are now proposed to provide an accurate identification of streptococcal isolates. Among these, restriction fragment length polymorphism analysis was proposed as a general method for bacterial identification and typing (15). Amplified ribosomal DNA (rDNA) restriction analysis (ARDRA) was recently reported to be a rapid and efficient method of identification of bacterial isolates to the species level in mollicutes (7) and in some genera such as Acinetobacter or Mycobacterium (30, 31). The initial protocol was substantially improved by amplification of a larger fragment of the rrn operon (16S rRNA gene, 16S-23S intergenic spacer, and 23S rRNA gene) followed by the digestion with one to three endonucleases selected according to the bacterial genus studied (12, 18). The latest modification of this method uses a unique combination of three restriction endonucleases (HhaI, MboII, and Sau3A) irrespective of the bacterial species studied. This modified protocol was successfully used for identification of the genus and the species of strains belonging to the family Enterobacteriaceae (G. Giammanco, P. Nogueira, O. Bouallegue, F. Grimont, and P. A. D. Grimont, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. R20, p. 482, 1998).

In the present study, we investigate the interest of the modified rrn-ARDRA protocol for identification of streptococci. One hundred and forty-eight strains were amplified and analyzed with HhaI, MboII, and Sau3A endonucleases. A database suitable for identification of streptococcal species was constructed and used to identify some clinical isolates.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains. A total of one hundred and forty-eight reference strains were included in the study (Table 1). These strains were obtained from the Australian Collection of Microorganisms (ACM), St. Lucia, Australia; the bioMérieux collection of microorganisms (API), La-Balme-les-Grottes, France; the American Type Culture Collection (ATCC), Manassas, Va.; the Culture Collection of the University of Göteborg (CCUG), Göteborg, Sweden; the Centers for Disease Control and Prevention (CDC), Atlanta, Ga.; the Collection de l'Institut Pasteur (CIP), Paris, France; le Centre National de Référence des Pneumocoques (CNRP), Créteil, France; the National Collection of Dairy Organisms (NCDO), Sheffield, United Kingdom; the National Collection of Type Cultures (NCTC), London, United Kingdom; and other depositors. Strains were cultured onto blood Columbia agar plates or brain heart infusion (BHI) broth medium (bioMérieux, La Balme-les-Grottes, France) and conserved at -80°C in BHI broth supplemented with glycerol (15%, vol/vol). For pneumococci, BHI broth was supplemented with 5% bovine ascite-serum (Bio-Rad, Marnes-la-Coquette, France), and cultures were incubated in a 5% CO2 atmosphere.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Identification, source, and restriction patterns of streptococcal strains studied for construction of database

 
Preparation of DNA. Bacteria (10 ml of a mid-log-phase culture) were harvested by centrifugation and washed twice in TE buffer (10 mM Tris, 1.0 mM Na2EDTA [pH 8.0]). Pellets were suspended in 500 µl of TES buffer (10 mM Tris, 1.0 mM Na2EDTA, 1 M sucrose [pH 8.0]) for lysis. Mutanolysin (5 U/ml; Sigma, St. Louis, Mo.) and lysozyme (10 mg/ml; Boehringer, Mannheim, Germany) were added, and the mixture was incubated overnight at 37°C. Bacterial membrane disruption was then done with proteinase K (0.4 mg/ml) and sodium dodecyl sulfate (1%, vol/vol). High-molecular-weight bacterial genomic DNA was purified from lysates by two sequential phenol-chloroform extraction and ethanol precipitation steps. DNA was dissolved in a buffer containing 10 mM Tris, 1 mM EDTA, and RNase (20 mg/ml). Its concentration and its integrity were determined visually after agarose (Bioprobe) gel electrophoresis (0.8%, wt/vol; 100 V; 1 h). Gels were stained by immersion in a 50-µg/ml ethidium bromide solution. DNA samples were diluted to a concentration of 1 µg/µl and microdialyzed (pore size, 0.22 µm; Millipore) before amplification.

Amplification. Oligonucleotide primers Ad (5'-AGAGTTTGATCMTGGCTCAG) and O24/3 (5'-CGACATCGAGGTGCCAAA) were derived from conserved regions present at the 5' end of the 16S rRNA gene and the 3' end of the 23S rRNA gene, respectively (Giammanco et al., Abstr. 98th Gen. Meet. Am. Soc. Microbiol.). Purified DNA (1 µg) was added to a 74-µl aliquot of PCR mixture containing 0.4 pmol (each) of primers Ad and O24/3 (Genset, Paris, France), 250 µmol (each) of deoxynucleoside triphosphate (Pharmacia Biotech, Orsay, France), and 0.05 U of Taq polymerase (Gibco BRL, Cergy Pontoise, France) in 1x reaction buffer. Magnesium chloride (1.1 mM; Gibco BRL), 0.05% (vol/vol) wetting agent (Gibco BRL), and 0.08% (wt/vol) bovine serum albumin (Sigma) were added to the mixture. Amplification was done in a Perkin-Elmer 2400 system as follows: initial denaturation at 95°C for 5 min, 30 cycles of denaturation at 94°C for 45 s and annealing at 60°C for 60 s, and extension at 72°C for 3 min. Finally, a 7-min extension period at 72°C was carried out. Positive (DNA from S. agalactiae CNRP 96/409) and negative controls were included in each run of amplification. The presence of a specific PCR product was controlled by agarose gel electrophoresis (0.8%, wt/vol; 100 V; 1 h). The molecular weight of the amplification product was calculated using a 1-kb marker from Sigma.

Restriction endonuclease digestion and analysis. Restriction was carried out for a 4-h period at 37°C in a 20-µl mixture containing 2 U of restriction enzymes HhaI, MboII, and Sau3AI (Amersham), 2 µl of the supplied specific incubation buffer, and 10 to 16 µl of PCR product. The volume of PCR product used in the restriction mixture was adjusted on the basis of the fluorescence intensity of the observed fragment in the control gel. Restriction was stopped by addition of 5 µl of 1x TE buffer (glycerol [50%, vol/vol], bromophenol blue 0.07%, wt/vol]). Before electrophoresis, restriction endonuclease digestions were heat inactivated as recommended by Giammenco et al. (14). Restriction fragment patterns were analyzed by gel electrophoresis at 120 V for 5 to 6 h in a 1.5% (wt/vol) Metaphor agarose (FMC BioProduct, Rockport, Maine) and 1.5% (wt/vol) standard agarose (Bioprobe) gel in 0.5x TAE buffer. DNA ladder 50-2000 (Bio-Rad) was loaded in four lanes of each gel for an accurate determination of the molecular weights of the restriction fragments. After migration, gels were stained by immersion in a 50-µg/ml BET solution for 1 h. Gels were then photographed and video acquired using Visiomic software (Genomic, Grenoble, France). The produced TIFF-formatted file was then reopened with the RestrictoScan program from the Taxotron package (Institut Pasteur, Paris, France). In each lane, restriction fragments were detected and their positions were consecutively noted. Calculation of the molecular size of each fragment was done using the Schaffer and Sederoff algorithm implemented in RestrictoTyper program. The sizes of the fragments were then entered in a database. Clustering of restriction patterns based on the single-linkage, average-linkage, and unweighted pair-group method using arithmetic averages (UPGMA) algorithms were done using the Adanson and Dendrograph programs from the Taxotron package.

In silico analysis. In order to test the reliability of our method, virtual restriction analyses were performed on the nucleotide sequence of the rrn operon from S. pyogenes ATCC 700294 (serotype M1). The sequences were retrieved from the S. pyogenes sequence data published by the University of Oklahoma Health Sciences Center (28). In silico analysis was performed using the Geneman program from the Lasergene software (DNAStar, Madison, Wis.). Results were compared to those provided by the experimental restriction patterns of the amplified rrn operon of S. pyogenes HDP 89539T (serotype M1), HDP 93106 (serotype M3), and HDP 93112 (serotype M48).

Identification of atypical streptococcal isolates. Eleven strains recently isolated from clinical specimens were studied. These isolates were identified as streptococci with conventional methods based upon morphological, cultural, and biochemical characteristics but exhibited either unexpected serological markers by the agglutination assay (Bio-Rad) or atypical biochemical traits when the rapid ID32STREP galleries (bioMérieux) were used. The three restriction patterns were first compared to their respective data base but, in an effort to allow a definitive identification, the three data bases were combined using the average-linkage algorithm. Restriction patterns of unknown isolates were then simultaneously compared to the complete data base, and a bacterial identification was then proposed. When analysis of the restriction patterns allowed identification of species represented in the library by a small number of strains, we confirmed the rrn-ARDRA identification by 16S rDNA sequence analysis as previously described (26).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Amplification and restriction of rrn operon. We have evaluated a total of 148 strains representative of 38 streptococcal species or subspecies mostly isolated from humans or animals. The PCR-based protocol allowed production of a single amplification product with an estimated size between 4,400 and 4,500 bp. This size was consistent with the size predicted after analysis of the published sequence of S. pyogenes ATCC 700294 (28). Electrophoretic analysis of the amplified product digested with HhaI, MboII, and Sau3A separately disclosed 7 to 14 bands ranging from 30 up to 1,500 bp (Fig. 1). BET staining and densitometric detection of smaller bands (i.e., bands under 100 bp) were not sufficiently reliable between runs, and these bands were not recorded for further analysis.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 1. Restriction analysis of amplifed operon with HhaI, MboII, and Sau3A restriction endonucleases. 1, S. pyogenes HDP 89539T; 2, S. infantarius HDP 90056T; 3, S. equinus HDP 89506T; 4, S. porcinus HDP 90049T; 5, molecular size marker; 6, S. pneumoniae HDP 89540T; 7, S. pyogenes HDP 93110; 8, S. constellatus HDP 89579T. (A) Visualization of the bands obtained after migration and BET staining. (B) Schematic representation of agarose gel. Only bands ranging from 100 to 2,000 bp are used for identification and are represented here.

 
In silico restriction analysis. Since the published sequence of the entire chromosome of S. pyogenes ATCC 700294 includes six amplified fragments of the rrn operon, we considered different sequences corresponding to positions 268.758 to 273.233 (4,476 bp), 515.696 to 520.172 (4,477 bp), 1.587.525 to 1.583.047 (4,479 bp), 1.772.590 to 1.768.155 (4,476 bp), 1.828.809 to 1.824.334 (4,475 bp), and 1.834.809 to 1.830.335 (4,475 bp). Each fragment was virtually digested with the three endonucleases. The six restriction patterns were then combined together and compared with the experimental patterns obtained with S. pyogenes HDP 89539T, HDP 93106, and HDP 93112. The real patterns displayed all the predicted bands and also few extra bands after HhaI and Sau3A digestion. However, these additional bands are very faint and may correspond to strain-specific alterations of the DNA sequence of only one or a few copies of the operon.

Restriction length polymorphism analysis. Visual observation of restriction patterns demonstrated a high interspecies variability and a low intraspecies variability (Fig. 1 and 2). In some species, the patterns successively obtained with the three restriction endonucleases were very homogeneous even though a large set of strains was studied (S. agalactiae, S. equi, S. gordonii, S. parauberis, S. uberis, and S. pyogenes strains). Thus, the construction of a similarity matrix and the statistical clustering of patterns improved the detection of small alterations in patterns obtained for strains of the same species. A computerized database was then built with restriction patterns using the average-linkage algorithm. The fragment size tolerance (experimental error) was set to 5%, which is superior to the usual interrun variations. Results of clustering analysis were identical when carried out by using UPGMA, single-linkage, or average-linkage analysis (data not shown). Initially the clustering analysis was done with each endonuclease separately, and a designation was assigned to each pattern, corresponding to a capital letter for major subdivisions of the dendrogram (distance between patterns > 0.25), an Arabic number for the different cluster of patterns inside these subdivisions (distance between 0.25 and 0.05), and a lowercase letter for minor variations of pattern (distance between patterns < 0.05) (Table 1).



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 2. Diagrammatic representation showing restriction patterns database for rrn-ARDRA obtained with type strains of major streptococcal species. Each restriction pattern was named separately in each restriction endonuclease database. The designation was assign to each digestion pattern corresponding to a capital letter for major subdivisions of the dendrogram (similarity index over 0.75), an arabic number for the different cluster of patterns inside of these subdivisions (similarity index between 0.75 and 0.95) and a lowercase letter for minor variations of pattern (similarity of patterns comprised between 0.95 and 0.99). Dendrogram was calculated using the UPGMA algorithm, for a database limited to the restriction pattern of the type strains of the species.

 
HhaI analysis alone allowed the delineation of most of the species, except those within the "mitis" group, including S. mitis, S. oralis, and S. pneumoniae (cluster designated as H-A with large strain-specific variations). Patterns obtained for S. bovis and S. gallolyticus isolates are also combined together (H-B3). Subspecies of S. dysgalactiae and S. equi were not delineated after digestion with HhaI endonuclease (patterns H-F2). Contrarily, restriction patterns of strains belonging to S. anginosus were assigned to different clusters (H-C1, H-E3, H-F3, and H-F4).

In the MboII database, all S. anginosus strains were grouped together (M-B5 patterns). Restriction patterns obtained with S. dysgalactiae subsp. equisimilis, S. dysgalactiae subsp. dysgalactiae, and S. canis were also merged (M-A3). At the same time, subspecies of S. equi were not distinguishable (patterns M-B6).

Patterns obtained with Sau3A displayed a large variability among S. pyogenes or S. porcinus strains, which were divided in two or three clusters (S-Q1, -Q2, and -Q3; S-K1 and -K2). Within S. dysgalactiae, two clusters of patterns were observed (S-R1 and -R2) irrespective of the subspecies delineation according to reference identification. In contrast to HhaI and MboII, the patterns obtained with Sau3A for the two subspecies S. equi subsp. equi and S. equi subsp. zooepidemicus were separated in two different clusters (S-N1 and S-E1, respectively). Restriction patterns obtained for oral streptococci belonging to the mitis group (S. mitis, S. oralis, and S. pneumoniae) were not grouped in the same branch of the dendrogram as was observed with HhaI or MboII endonuclease.

The simultaneous observation of restriction patterns obtained with the three endonucleases was then proposed to improve the differentiation of strains corresponding to the different species and subspecies (Fig. 1 and 2). Minor alterations observed in one of the restriction patterns (band shift or addition or suppression of a single band) have a limited effect upon clustering when the two patterns obtained with the other restriction endonucleases are similar. At the same time, strains belonging to different subspecies and distinguished only by patterns obtained with one endonuclease were separated in the cumulative database. All the strains belonging to the 36 species studied, including type strains, reference strains, and clinical isolates, were included in separated clusters. The phylogenic tree obtained for cumulative analysis of the three sets of data after UPGMA analysis of type strains is presented in Fig. 2. A distance index of <0.05 was found for four pairs of related streptococcal species: S. equinus and S. bovis, S. gallolyticus and S. caprinus, S. agalactiae and S. difficilis, and S. alactolyticus and S. intestinalis, respectively. These results suggest that each of these couples corresponds to a single species. S. dysgalactiae subsp. dysgalactiae, S. dysgalactiae subsp. equisimilis, and S. canis appeared close to each other in cumulative analysis. In contrast, restriction patterns obtained for S. equi subsp. equi and S. equi subsp. zooepidemicus were clearly separated.

Identification of clinical isolates. A total of 11 streptococcal strains identified with atypical characteristics were subjected to rrn-ARDRA analysis. Biochemical identification was confirmed in three cases and changed to another species in seven cases (Table 2). In four cases, the rrn-ARDRA identification was confirmed by the 16S rDNA analysis, including one strain which was initially unidentified according to its biochemical patterns and assigned to S. anginosus.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Identification of atypical or phenotypically unidentified streptococcal strains by rrn-ARDRA

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Among molecular tools of bacterial identification, the determination of rRNA gene restriction patterns (ribotyping) has been extensively used (15). However, this methodology requires several steps, including transfer onto membranes and hybridization steps. Therefore, some alternative techniques have been proposed in an effort to obtain faster results. PCR amplification of DNA followed by restriction analysis of amplification products has attracted a growing interest. ARDRA (i.e., amplification and polymorphism of restriction analysis of 16S rRNA genes) has been proposed for the identification of species within some genera, such as Acinetobacter, Mycobacterium, and Mycoplasma (7, 30, 31). However, analysis restricted to the 16S rRNA gene appeared to be of limited value in some other genera (18). It was then suggested that inclusion of the 23S rRNA gene and 16-23S spacer in the analysis may improved the differentiation between species in some genera (18; Giammanco et al., Abstr. 98th Gen. Meet. Am. Soc. Microbiol.) as it allows the study of a larger amount of genetic information. Indeed, due to evolutionary constraints, there is a minimal variation into the 16S or the 23S rRNA sequences, whereas the 16S-23S intergenic sequence demonstrated an enhanced variability. The similarity of the sequences of 16S rRNA gene between different streptococcal species was determined to be between 91 and 99% (1). In contrast, the 16S-23S rRNA internal spacer is subjected to a minimal selective pressure compared to rRNA genes. Insertion, deletion, and single-base changes are frequent in this DNA fragment, and sequence identity between the intergenic spacers of different streptococcal species is limited to between 50 and 80% (10). In our study, a single band corresponding to the amplification of the rrn operon of streptococci was observed on the electropherogram. This indicates that the potential difference of lengths between the rrn operons of distinct species or between the six different rrn operons of the streptococcal strains (28) remains within the resolution of the agarose gel we used.

To determine the advantage and the reliability of the improved rrn-ARDRA protocol of identification, we have investigated 148 type and reference strains representative of the 39 major species and subspecies isolated from humans or animals. All these strains have been phenotypically characterized by their initial depositor, and most of them have also been identified by DNA-DNA hybridizations. A large interspecific variability of restriction profiles was detected among the species studied. When available, three to seven reference strains of each species were included in the study. The results of these investigations suggest that restriction patterns were conserved within species. So, restriction patterns obtained with strains of S. pyogenes were homogeneous and similar to the profiles predicted by DNA sequence analysis. In fact, controversial data were published about the presence or the absence of variable restriction sites in different copies of the 16S rRNA gene within the bacterial chromosome. Some studies reported the absence of intraspecies variations of 16S rRNA genes (18, 30), but a large variability of restriction patterns of 16S RNA was observed within Abiotrophia adiacens in the study of a large collection of Abiotrophia strains (23). Gurtler et al. (16) have also reported highly variable restriction patterns in a recent analysis of the clostridial species. Alterations of one or a few copies of the rrn operon may have resulted in faint bands on the electropherogram, but these additional bands, like the bands due to minor or incomplete digestion, did not appear essential for the differentiation between the restriction patterns of different species (31). Some other minor variations of restriction patterns appeared unpredictable according to the species studied or to the restriction endonuclease used. These variations were assumed to be strain specific.

The construction of a combined database using the three restriction patterns obtained separately for each strain was proposed to overtake the strain variability and achieve a better delineation of species (Giammanco et al., Abstr. 98th Gen. Meet. Am. Soc. Microbiol.). Using the triple combination of HhaI, MboII, and Sau3A restriction endonucleases, strains belonging to the same species are found to exclusively cluster in the same restriction patterns. When 3 to 10 strains were studied, none of the strains were assigned to an inadequate cluster with the noticeable exceptions of S. dysgalactiae and S. canis, as well as S. pneumoniae, S. mitis, and S. oralis. We observed that enlargement of the available database of patterns usually induces a better clustering of the restriction patterns around the type strain of the species. The dendrogram that we obtained using average-linkage or UPGMA clustering methods confirms the major subdivisions of the classification of streptococci and the results based on rRNA (1) or superoxide dismutase (19, 24) sequence analysis

The classification of S. anginosus, S. constellatus, and S. intermedius, which has now been clarified (33), is clearly delineated using rrn-ARDRA. However, the similarity of their HhaI and Sau3A patterns confirms the phylogenic proximity of these three species (33). In the case of S. uberis and S. parauberis, where biochemical or serological differentiation is not possible (2, 34), rrn-ARDRA clearly distinguishes between the two species. We also noticed the inclusion of strains belonging to the species S. bovis, S. caprinus, S. difficilis, or S. intestinalis into the respective clusters of S. equinus, S. gallolyticus, S. agalactiae, and S. alactolyticus. Our data appear consistent with the results of DNA-DNA reassociation experiments which have recently demonstrated the synonymy of these species.

The strains of S. porcinus, S. pyogenes, or S. pneumoniae cluster together within their respective species, including whatever Lancefield's serogroups or serotypes they belong, allowing a rapid species identification. Since similar conserved patterns were observed for different strains within the same species, rrn-ARDRA may have a reduced epidemiological (typing) interest. rrn-ARDRA could not differentiate between the two subspecies of S. dysgalactiae and S. canis. These species are part of a large and heterogeneous group of C, G, or L streptococci associated with human and animal infections. HhaI, MboII, and Sau3A patterns are quite similar for these three species as well, although DNA homology between species is between 45 and 75% (32) and 16S rRNA similitude value is limited to 96 to 97% (1). However, the classification of this group remained controversial as no test was clearly capable of differentiating these species (9, 29, 32).

Similarly, identification of strains belonging to S. pneumoniae, S. oralis, and S. mitis to the species level is also difficult with rrn-ARDRA. These three species constitute a single cluster of restriction patterns and the distinct differences seen between the three type strains are not observed with other species. Similar results were found using analysis of superoxide dismutase (19, 24) or the 16S rRNA gene sequence (1). S. pneumoniae, S. oralis, and S. mitis were demonstrated to form a bacterial complex in which both biochemical or genotypical differentiation may be difficult (K. Poulsen and M. Kilian, ASM Conf. Streptococcal Genet., abstr. 2D-05, 1998). In contrast, species belonging to the streptococcal group of S. sanguinis, S. parasanguinis, and S. gordonii displayed restriction patterns clearly separated from the S. pneumoniae, S. oralis, and S. mitis complex.

The data presented here show that PCR amplification followed by restriction analysis of the amplified products is a reliable and easy-to-use method of identification of streptococci. It has been demonstrated on a limited scale to be capable of identifying clinical isolates that were phenotypically mis- or not identified and appears to be a promising method for identification of pathogenic streptococci.

As observed for identification of Enterobacteriaceae or Acinetobacter, the performances of rrn-ARDRA were found excellent for identification of gram-positive, catalase negative cocci belonging to the genus Streptococcus. In order to improve these performances, determination of restriction patterns of large set of bacteria have to be conducted and databases of patterns have to be extended. Using our modified rrn-ARDRA protocol, a single database which combine the three restriction patterns obtained for each strain, may allow a rapid identification irrespective of the Gram staining or the morphological characteristics of the isolate. We propose to use this method in reference centers for identification of phenotypically unidentified isolates after construction and evaluation of specific databases of interest.


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre National de Référence des Streptocoques, Service de Microbiologie, Hôtel Dieu, Université Paris VI, 1, place du Parvis Notre-Dame, F-75181 Paris cedex 04, France. Phone: 33 1 42 34 82 73. Fax: 33 1 42 34 87 19. E-mail: anne.bouvet{at}htd.ap-hop-paris.fr. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. 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]
  2. Bentley, R. W., J. A. Leigh, and M. D. Collins. 1993. Development and use of species-specific oligonucleotide probes for differentiation of Streptococcus uberis and Streptococcus parauberis. J. Clin. Microbiol. 31:57-60.[Abstract/Free Full Text]
  3. Bouvet, A., F. Grimont, and P. A. D. Grimont. 1991. Intraspecies variations in nutritionally variant streptococci: rRNA gene restriction patterns of Streptococcus defectivus and Streptococcus adjacens. Int. J. Syst. Bacteriol. 41:483-486.[Abstract/Free Full Text]
  4. Bouvet, A., F. Grimont, M. D. Collins, F. Benaoudia, C. Devine, B. Regnault, and P. A. D. Grimont. 1997. Streptococcus infantarius sp. nov. related to S. bovis and S. equinus. Adv. Exp. Med. Biol. 418:393-395.[Medline]
  5. Bridge, P. D., and P. H. A. Sneath. 1983. Numerical taxonomy of Streptococcus. J. Gen. Microbiol. 129:565-597.[Medline]
  6. Coykendall, A. L. 1989. Classification and identification of the viridans streptococci. Clin. Microbiol. Rev. 2:315-328.[Abstract/Free Full Text]
  7. Deng, S., C. Hiruki, J. A. Robertson, and G. W. Stemke. 1992. Detection by PCR and differentiation by restriction length polymorphism of Acholeplasma. Spiroplasma, Mycoplasma, and Ureaplasma, based upon 16S rRNA genes. PCR Methods Appl. 1:202-204.[Medline]
  8. Doit, C., F. Grimont, R. A. Whiley, B. Regnault, P. A. D. Grimont, J. M. Hardie, and A. Bouvet. 1994. Ribotypes of the ‘Streptococcus milleri'-group allow discrimination between strains of Streptococcus constellatus, Streptococcus intermedius and Streptococcus anginosus, p. 531-532. In A. Totolian (ed.), Pathogenic streptococci, present and future. Lancer. St. Petersburg, Publications, Russia.
  9. Efstratiou, A., G. Colman, G. Hahn, J. F. Timoney, J. M. Bouefgras, and D. Monget. 1994. Biochemical differences among human and animal streptococci of Lancefield group C or group G. J. Med. Microbiol. 41:145-148.[Abstract]
  10. Forsman, P., A. Tilsala-Timisjarvi, and T. Alatossava. 1997. Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S-23S rRNA spacer regions. Microbiology 143:3491-3500.[Abstract]
  11. Freney, J., S. Bland, J. Etienne, M. Desmonceaux, J. M. Boeufgras, and J. Fleurette. 1992. Description and evaluation of the semiautomated 4-hour rapid ID32Strep method for identification of streptococci and members of related genera. J. Clin. Microbiol. 30:2657-2661.[Abstract/Free Full Text]
  12. Garcià-Arata, M. I., P. Gerner-Smidt, F. Baquero, and A. Ibrahim. 1997. PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level. Res. Microbiol. 148:777-784.[Medline]
  13. Garnier, F., G. Gerbaud, P. Courvalin, and M. Galimand. 1997. Identification of clinical relevant viridans group streptococci to the species level by PCR. J. Clin. Microbiol. 35:2337-2341.[Abstract]
  14. Giammanco, G. M., F. Grimont, and P. A. D. Grimont. 1999. MboII endonuclease heat inactivation before agarose gel electrophoresis to prevent artifactual bands in restriction patterns. BioTechniques 27:886-887.[Medline]
  15. Grimont, F., and P. A. D. Grimont. 1986. Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Ann. Inst. Pasteur Microbiol. B 137:165-175.
  16. Gurtler, V., V. A. Wilson, and B. C. Mayall. 1991. Classification of medically important clostridia using restriction endonuclease site difference of PCR-amplified 16S rDNA. J. Gen. Microbiol. 137:2673-2679.[Medline]
  17. Hardie, J. M. 1986. Genus Streptococcus, p. 1043-1071. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore, Md.
  18. Ibrahim, A., P. Gerner-Smidt, and S. Sjöstedt. 1996. Amplification and restriction endonuclease digestion of a large fragment of genes coding for rRNA as a rapid method for discrimination of closely related pathogenic bacteria. J. Clin. Microbiol. 34:2894-2896.[Abstract]
  19. Kawamura, Y., R. A. Whiley, S. E. Shu, T. Ezaki, and J. Hardie. 1999. Genetic approaches to the identification of the mitis group within the genus Streptococcus. Microbiology 145:2605-2613.[Abstract/Free Full Text]
  20. Kilian, M., L. Mikkelsen, and J. Henrichsen. 1989. Taxonomic study of viridans streptococci: description of Streptococcus gordonii sp. nov. and emended description of Streptococcus sanguis (White and Niven 1946). Streptococcus oralis (Bridge and Sneath 1982) and Streptococcus mitis (Andrewes and Horder 1906). Int. J. Syst. Bacteriol. 39:417-484.
  21. Kilpper-Bälz, R., and K. H. Schleifer. 1984. Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci. FEMS Microbiol. Lett. 24:355-364.[CrossRef]
  22. Lancefield, R. C. 1933. A serological differentiation of human and other groups of hemolytic streptococci. J. Exp. Med. 59:571-591.
  23. Ohara-Nemoto, Y., S. Tajika, M. Sasaki, and M. Kaneko. 1997. Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis. J. Clin. Microbiol. 35:2458-2463.[Abstract]
  24. Poyard, C., G. Quesne, S. Coulon, P. Berche, and P. Trieu-Cuot. 1998. Identification of streptococci to species level by sequencing the gene encoding the manganese-dependant superoxide dismutase. J. Clin. Microbiol. 36:41-47.[Abstract/Free Full Text]
  25. Rudney, J. D., and C. J. Larson. 1993. Species identification of oral viridans streptococci by restriction fragment polymorphism analysis of rRNA genes. J. Clin. Microbiol. 31:2467-2473.[Abstract/Free Full Text]
  26. Schlegel, L., F. Grimont, M. D. Collins, B. Régnault, P. A. D. Grimont, and A. Bouvet. 2000. Streptococcus infantarius sp. nov., Streptococcus infantarius subsp. infantarius subsp. nov., and Streptococcus infantarius subsp coli subsp. nov., isolated from humans and food. Int. J. Syst. Evol. Microbiol. 50:1425-1434.[Abstract]
  27. Schleifer, K. H., and R. Klipper-Bälz. 1987. Molecular and chemotaxonomic approches to the classification of streptococci, enterococci and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
  28. Suvorov, A. N., and J. J. Ferretti. 1996. Physical and genetic chromosomal map of an M-type 1 strain of Streptococcus pyogenes. J. Bacteriol. 178:5546-5549.[Abstract/Free Full Text]
  29. Vandamme, P., B. Pot, E. Falsen, K. Kersters, and L. A. Devriese. 1996. Taxonomic studies of Lancefield streptococcal group C, G, and L (Streptococcus dysgalactiae) and proposal of S. dysgalactiae subsp. equisimilis subsp. nov. Int. J. Syst. Bacteriol. 46:774-781.[Abstract/Free Full Text]
  30. Vaneechoutte, M., H. De Beenhouwer, C. Claeys, G. Verschraegen, A. De Rouck, N. Paepe, A. Elaichouni, and F. Portaels. 1993. Identification of Mycobacterium species with amplified rDNA restriction analysis. J. Clin. Microbiol. 31:2061-2065.[Abstract/Free Full Text]
  31. Vaneechoutte, M., L. Dijkshoorn, I. Tjernberg, A. Elaichouni, P. De Vos, C. Claeys, and G. Verschraegen. 1995. Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J. Clin. Microbiol. 33:11-15.[Abstract]
  32. Vieira, V. V., L. M. Teixera, V. Zahner, H. Momen, R. R. Facklam, A. G. Steigerwalt, D. J. Brenner, and A. C. D. Castro. 1998. Genetic relationships among the different phenotypes of Streptococcus dysgalactiae strains. Int. J. Syst. Bacteriol. 48:1231-1243.[Abstract/Free Full Text]
  33. Whiley, R. A., and D. Beighton. 1991. Emended description and recognition of Streptococcus constellatus, Streptococcus intermedius and Streptococcus anginosus as distinct species. Int. J. Syst. Bacteriol. 41:1-5.[Abstract/Free Full Text]
  34. Williams, A., and M. D. Collins. 1990. Molecular taxonomic studies on Streptococcus uberis types I and II. Description of Streptococcus parauberis sp. nov. J. Appl. Bacteriol. 68:485-490.

Journal of Clinical Microbiology, February 2003, p. 657-666, Vol. 41, No. 2
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.2.657-666.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schlegel, L.
Right arrow Articles by Bouvet, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schlegel, L.
Right arrow Articles by Bouvet, A.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS