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Journal of Clinical Microbiology, December 2008, p. 3875-3879, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00810-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene

Tomasz Hauschild^1* and Srdjan Stepanovi²

Department of Microbiology, Institute of Biology, University of Bialystok, Bialystok 15-950, Poland,¹ Department of Bacteriology, Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Belgrade 11000, Serbia²

Received 29 April 2008/ Returned for modification 2 May 2008/ Accepted 23 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

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Staphylococci are widely distributed in various environments. Natural populations are associated with the skin, skin glands, and mucous membranes of humans and many animals alike. They are sometimes found in the intestinal, genitourinary, and upper respiratory tracts of these hosts. They have also been isolated from animal products and other sources, such as soil, sand, seawater, fresh water, dust, and air (9). So far, 40 species of the genus Staphylococcus have been identified (excluding Staphylococcus pulvereri as a separate species), 10 of which contain subdivisions with subspecies designations.

Although Staphylococcus aureus is clinically the most significant Staphylococcus species, as it causes many types of infections, other coagulase-negative staphylococci (CoNS) are increasingly becoming recognized as etiologic agents of medical device-related infections in humans, as well as different types of infections in farm and pet animals. Consequently, it is becoming increasingly important to accurately identify these isolates to the species level in order to define the clinical significance of the bacteria in question, to carry out proper epidemiologic observations, and to manage CoNS-infected patients with relapses. A variety of manual and automated methods based on phenotypic characteristics have been developed for the identification of staphylococci, including conventional identification methods and several methods that use commercial kits. Unfortunately, the overall accuracies of these systems are low and range from 50 to 70% (6, 8, 15, 16). Moreover, conventional reference methods are too laborious and time-consuming to be used in clinical laboratories. Several problems associated with the systems mentioned above result from the variability in the expression of metabolic activities and/or the morphological features of some staphylococcal species (4); thus, if the strain has atypical characteristics, it may be difficult, if not impossible, to precisely assign the strains to the species level. Furthermore, commercial systems may offer two or more suggestions as to the species identification with comparable levels of safety. Due to the limited number of stable features that can be used for species discrimination, many taxa remain difficult to distinguish from one another and are misidentified by phenotypic tests (4). To solve these problems, restriction fragment length polymorphism (RFLP) analysis of PCR products and a number of PCR amplicon sequencing-based methods have been reported for use for the identification of staphylococci (1, 2, 5, 10, 11, 12, 13, 14, 17, 18, 21, 22, 23, 24, 25).

This paper describes a sensitive and specific nucleic acid-based procedure that is able to differentiate 41 Staphylococcus species and subspecies, based on PCR-RFLP analysis of the dnaJ gene.

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Bacterial strains. The bacterial strains used in this study are described in Table 1. The 47 reference Staphylococcus species and subspecies were selected from the Polish Collection of Microorganisms, the Czech Collection of Microorganisms, and the American Type Culture Collection. The clinical isolates consisted of five S. aureus strains, seven S. haemolyticus strains, three S. epidermidis strains, four S. sciuri strains, one S. vitulinus strain, one S. caprae strain, one S. cohnii subsp. cohnii strain, and one S. hominis strain that were previously identified by use of the BD Phoenix system or reference biochemical tests (3, 20).

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TABLE 1. Staphylococcal reference strains and clinical isolates used in this study

Chromosomal DNA isolation. Chromosomal DNAs from all staphylococcal strains were obtained from overnight cultures grown in 3 ml of brain heart infusion broth. After centrifugation of 0.5 ml of culture at 4,000 x g for 10 min, the bacterial pellet was suspended in 100 µl of lysis buffer (20 mM Tris-HCl [pH 8.3], 10 mM EDTA [pH 8.3], 10 mM NaCl, 5 µl [1 mg/ml] of lysostaphin) and the mixture was incubated for 1 h at 37°C. The samples were then incubated for 10 min at 95°C. After a 10-min centrifugation of the mixture at 12,000 x g, the chromosomal DNAs in the supernatants were used for PCR analysis.

16S rRNA gene analysis. Amplification of an 880-bp part of the 16S rRNA gene from chromosomal DNA was achieved by PCR with forward primer SSU-bact-27f (5'-AGA GTT TGA TCM TGG CTC AG-3') and reverse primer SSU-bact-907r (5'-CCG TCA ATT CMT TTR AGT TT-3'), which have been described previously (2). The thermal cycling conditions were 5 min at 94°C for 1 cycle, followed by 30 cycles of 45 s at 94°C, 1 min at 55°C, and 1.5 min at 72°C. The last cycle was performed for 10 min at 72°C. The broad-range primers SSU-bact-27f and SSU-bact-519r (5'-GWA TTA CCG CGG CKG CTG-3') were used for sequencing of both strands of the 5' ends of the 16S rRNA genes. Briefly, sequencing was performed with a total volume of 10 µl containing 0.5 µl of premix from the ABI Prism BigDye Terminator (version 3.0) ready reaction cycle sequencing kit (Applied Biosystems), 1.8 µl of buffer (400 mM Tris-HCl, 10 mM MgCl₂), 10 pmol of sequencing primer, and 2 µl of the cleaned PCR product. The sequencing products were purified with ExTerminator kits (A&A Biotechnology, Poland) and were analyzed on an ABI Prism 3130 genetic analyzer, as specified by the manufacturer (Applied Biosystems). For identification, the partial 16S rRNA gene sequences were compared with sequences from GenBank.

PCR-RFLP analysis of gap gene. For the identification of randomly chosen clinical isolates, PCR-RFLP analysis of the gap gene was carried out as described previously (24, 25).

PCR-RFLP analysis of dnaJ gene. The previously described (17) dnaJ degenerate primers SA-(F) (5'-GCC AAA AGA GAC TAT TAT GA-3') and SA-(R) (5'-ATT GYT TAC CYG TTT GTG TAC C-3') were used to amplify the dnaJ gene fragment. The PCR mixtures were first incubated for 3 min at 94°C, followed by five cycles at 94°C for 30 s, 45°C for 30 s, and 72°C for 60 s. The mixtures were then subjected to a series of 30 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s; these were completed with a final extension at 72°C for 3 min. The presence of a PCR product was confirmed by 1% (wt/vol) agarose gel electrophoresis and visualization with ethidium bromide.

On the basis of computational restriction fragment analysis of the partial dnaJ gene sequences (GenBank database accession nos. AB234056 to AB2343089, AB234320 to AB234329, and AB234319) with the Restriction Mapper program (version 3; http://www.restrictionmapper.org), the XapI or Bsp143I restriction enzyme was chosen, as these enzymes provide species-specific restriction profiles for the majority of the staphylococcal species and subspecies. Digestions were performed with 5 µl of the PCR products in a total volume of 15 µl with 1x reaction buffer and either 10 U of the XapI endonuclease or 10 U of the Bsp143I endonuclease (Fermentas, Lithuania) for 3 h at 37°C. The resulting fragments were separated by electrophoresis on a 2% TopVision agarose gel (Fermentas) and were visualized under UV light after ethidium bromide staining.

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The dnaJ gene was amplified by PCR with a Staphylococcus-specific pair of primers, the specificities of which have been examined previously (17). To see if the 47 reference Staphylococcus species and subspecies tested in this study could be differentiated, the 920-bp products amplified from these species and subspecies by PCR were digested with XapI, and the resulting fragments were separated by agarose gel electrophoresis (Fig. 1A and B). Thirty-five distinctive RFLP patterns were obtained for all staphylococcal reference strains. The subspecies of S. capitis, S. carnosus, S. cohnii, and S. hominis produced different RFLP patterns. However, it was not possible to differentiate between the (sub)species pairs S. auricularis and S. felis, S. caprae and S. pasteuri, S. carnosus subsp. carnosus and S. warneri, S. cohnii subsp. cohnii and S. epidermidis, S. kloosii and S. saprophyticus, and S. nepalensis and S. pettenkoferi or between subspecies of the species S. aureus, S. equorum, S. saprophyticus, S. sciuri, and S. succinus. Therefore, the 920-bp PCR-amplified products of (sub)species not discriminated by the first digestion were digested with Bsp143I, which clearly distinguished the different RFLP patterns (Fig. 2). This second single digestion allowed the discrimination of all subspecies except for those of S. aureus, S. equorum, S. saprophyticus, S. sciuri, and S. succinus. In this way, the combination of separate digestions of the dnaJ gene with two different restriction enzymes allowed the precise identification of the 41 Staphylococcus species and subspecies.

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FIG. 1. (A and B) Agarose gel electrophoresis of the fragments produced by XapI digestion of the PCR amplification products from Staphylococcus reference strains (Table 1). Lanes: 1, S. arlettae; 2, S. aureus subsp. anaerobius; 3, S. aureus subsp. aureus; 4, S. auricularis; 5, S. capitis subsp. capitis; 6, S. capitis subsp. urealyticus; 7, S. caprae; 8, S. carnosus subsp. carnosus; 9, S. carnosus subsp. utilis; 10, S. chromogenes; 11, S. cohnii subsp. cohnii; 12, S. cohnii subsp. urealyticus; 13, S. condimenti; 14, S. epidermidis; 15, S. equorum subsp. equorum; 16, S. equorum subsp. linens; 17, S. felis; 18, S. fleurettii; 19, S. gallinarum; 20, S. haemolyticus; 21, S. hominis subsp. hominis; 22, S. hominis subsp. novobiosepticus; 23, S. hyicus; 24, S. intermedius; 25, S. kloosii; 26, S. lentus; 27, S. lugdunensis; 28, S. muscae; 29, S. nepalensis; 30, S. pasteuri; 31, S. pettenkoferi; 32, S. piscifermentans; 33, S. pseudintermedius; 34, S. saccharolyticus; 35, S. saprophyticus subsp. bovis; 36, S. saprophyticus subsp. saprophyticus; 37, S. schleiferi subsp. coagulans; 38, S. sciuri subsp. carnaticus; 39, S. sciuri subsp. rodentium; 40, S. sciuri subsp. sciuri; 41, S. simulans; 42, S. succinus subsp. casei; 43, S. succinus subsp. succinus; 44, S. vitulinus; 45, S. warneri; 46, S. xylosus; 47, S. simiae; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).

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FIG. 2. Agarose gel electrophoresis of the fragments produced by Bsp143I digestion of the PCR amplification products from Staphylococcus reference strains (Table 1). Lanes: 1, S. auricularis; 2, S. felis; 3, S. caprae; 4, S. pasteuri; 5, S. carnosus subsp. carnosus; 6, S. warneri; 7, S. cohnii subsp. cohnii,; 8, S. epidermidis; 9, S. kloosii; 10, S. saprophyticus subsp. bovis; 11, S. nepalensis; 12, S. pettenkoferi; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).

A total of 23 clinical isolates of eight Staphylococcus species were tested to validate the results of PCR-RFLP analysis of the dnaJ gene in a clinical setting. All of these isolates were previously identified by use of the BD Phoenix system and conventional biochemical tests at the (sub)species level: five S. aureus isolates, one S. caprae isolate, one S. cohnii subsp. cohnii isolate, three S. epidermidis isolates, seven S. haemolyticus isolates, one S. hominis isolate, four S. sciuri isolates, and one S. vitulinus isolate (Table 1). To confirm the correct phenotypic identification of the clinical isolates, definitive species identification was achieved on the basis of both the sequence data for the 16S rRNA gene and PCR-RFLP analysis of the gap gene for the S. caprae, S. cohnii subsp. cohnii, S. hominis, and S. vitulinus isolates and one randomly chosen isolate of each of the species S. aureus, S. epidermidis, S. haemolyticus, and S. sciuri. The BD Phoenix system misidentified three clinical isolates. S. caprae, S. cohnii subsp. cohnii, and S. hominis were identified by both genotypic methods as S. haemolyticus, S. cohnii subsp. urealyticus, and S. epidermidis, respectively (Table 2). The genotypic identifications of the remaining clinical isolates were in conformity with the previous identifications made by the phenotypic methods. All clinical isolates definitively identified as S. aureus, S. cohnii subsp. urealyticus, S. epidermidis, S. haemolyticus, S. sciuri and S. vitulinus presented the same RFLP pattern by analysis of the dnaJ gene as the corresponding reference strains (Fig. 1A and B and Fig. 3), and comparison of the results of PCR-RFLP analysis of the dnaJ and gap genes and 16S rRNA gene sequencing showed that they were 100% concordant, as summarized in Table 2.

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TABLE 2. Phenotypic and genotypic identification of clinical isolates

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FIG. 3. Agarose gel electrophoresis of the fragments produced by XapI digestion of the PCR amplification products from clinical Staphylococcus isolates. Lanes: 1, S. aureus; 2, S. haemolyticus; 3, S. cohnii subsp. urealyticus; 4, S. epidermidis; 5, S. sciuri; 6, S. vitulinus; M, DNA molecular mass markers, 50-bp ladder (bottom band, 50 bp).

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The use of nucleic acid targets, with their high sensitivity and specificity, provides an alternative technique for the accurate identification and classification of Staphylococcus species. Gene sequence-based identification of bacteria to the species level may require resolution of the whole gene; in some cases, however, phylogenetically closely related bacterial species cannot be differentiated from one another. Although comparison of the 16S rRNA gene sequences has been useful in phylogenetic studies at the genus level, its use has been questioned in studies at the species level. This stems from the fact that closely related species may have identical 16S rRNA sequences or, alternatively, that divergent 16S rRNA sequences may exist within a single organism (19). To solve this problem, it is possible to use alternative monocopy target sequences which exhibit a higher degree of divergence than that of the 16S rRNA gene. Recently, partial sequencing of the highly conserved and ubiquitous hsp60, femA, tuf, sodA, rpoB, and dnaJ genes has been considered useful for the identification and taxonomic classification of species of the genus Staphylococcus (5, 11, 12, 13, 17, 18, 23). The dnaJ gene encodes the DnaJ protein, which is also known as Hsp40, and is a member of the heat shock protein (Hsp) family; and the dnaJ gene sequence is more discriminative than the sequences of other conserved, housekeeping genes used in the taxonomy of staphylococci. In addition, the evolutionary rate of substitution of the dnaJ sequence is much faster than that of the 16S rRNA gene sequence (17), and thus, the dnaJ gene sequence is potentially useful for the identification of genetically related species and even subspecies.

On the other hand, PCR-RFLP analysis is useful for the taxonomy of staphylococci and proves to be easier, less expensive, and less equipment dependent than sequencing. Recently, PCR-RFLP analysis of the internal transcribed spacer of the 16S rRNA gene, the intergenic spacer of the 16S-23S rRNA gene, the tuf gene, the gap gene, and the groEL gene has been developed for the identification of Staphylococcus species; but the number of species and subspecies that could be identified did not exceed 25, and these were mainly limited to the most common human and animal staphylococcal pathogens (1, 10, 14, 21, 25). The novel method based on PCR-RFLP analysis of the dnaJ gene described here is able to increase considerably the list of species that can be precisely identified to as many as 41, including species associated with human and animal infections that could not be classified by PCR-RFLP analysis of other genes. It is worth mentioning that this method is also able to discriminate subspecies of the species S. capitis, S. carnosus, S. cohnii, and S. hominis. However, the paucity of data on the divergence of the dnaJ sequence within staphylococcal species makes it difficult to state whether this technique would not suffer the same criticism over the accurate identification of staphylococci at the species level as it was in the case of phylogenetic studies based on 16S rRNA gene sequence analysis.

To validate the utility of PCR-RFLP analysis of the dnaJ gene as an identification method, the analysis was performed with a set of clinical isolates of eight Staphylococcus species. The data for 23 clinical isolates showed that (sub)species-specific RFLP patterns were observed. As part of the process of validation of PCR-RFLP analysis of the dnaJ gene, the results were compared with those obtained by PCR-RFLP analysis of the gap gene and 16S rRNA gene sequencing, and a 100% concordance of the results was shown when the results for the identification of S. cohnii by 16S rRNA gene sequencing and the identification of S. sciuri and S. vitulinus by PCR-RFLP analysis of the gap gene were excluded. Genotypic identification based on 16S rRNA gene sequences has limited discriminatory power for closely related Staphylococcus (sub)species, and RFLP patterns of the gap gene have never been detected for members of the S. sciuri group. Moreover, our results showed that the BD Phoenix system misidentifies S. hominis isolates, which was consistent with the results of comparative studies on the species-level identification of CoNS (7), and this system provided incorrect identifications for S. caprae and S. cohnii subsp. cohnii isolates.

In conclusion, the present study has demonstrated that PCR-RFLP analysis of the dnaJ gene with XapI or Bsp143I digestion proved to be an adequate tool for the correct identification of almost all prevalent species and subspecies of Staphylococcus, irrespective of their phenotypic characterization. This method requires only PCR and one or two enzymes and thus is technically less demanding than the majority of other molecular approaches. PCR-RFLP analysis might be useful not only in research laboratories but also in reference laboratories. Most importantly, the method under discussion allows the correct identification of staphylococcal strains that could not be assigned to a species level or that are misidentified as related staphylococci by phenotypic tests.

 
* Corresponding author. Mailing address: Department of Microbiology, Institute of Biology, University of Bialystok, Swierkowa 20 B, Bialystok 15-950, Poland. Phone: 48 85 74 57 428. Fax: 48 85 74 57 302. E-mail: thausch{at}uwb.edu.pl

Published ahead of print on 1 October 2008.

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1
1. Barros, E. M., N. L. Iório, C. Bastos Mdo, K. R. dos Santos, and M. Giambiagi-deMarval. 2007. Species-level identification of clinical staphylococcal isolates based on polymerase chain reaction-restriction fragment length polymorphism analysis of a partial groEL gene sequence. Diagn. Microbiol. Infect. Dis. 59:251-257.[Medline]
2
2. Becker, K., D. Harmsen, A. Mellmann, C. Meier, P. Schumann, G. Peters, and C. von Eiff. 2004. Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J. Clin. Microbiol. 42:4988-4995.[Abstract/Free Full Text]
3
3. Cirkovic, I., S. Stepanovic, D. Vukovic, M. Svabic-Vlahovic, V. Dimitrijeviæ, and T. Hauschild. 2008. Evaluation of the BD Phoenix automated microbiology system for detecting methicillin-resistant Staphylococcus aureus. Int. J. Antimicrob. Agents 32:94-95.[CrossRef][Medline]
4
4. Couto, I., S. Pereira, M. Miragaia, I. S. Sanches, and H. de Lencastre. 2001. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR. J. Clin. Microbiol. 39:3099-3103.[Abstract/Free Full Text]
5
5. Drancourt, M., and D. Raoult. 2002. rpoB gene sequence-based identification of Staphylococcus species. J. Clin. Microbiol. 40:1333-1338.[Abstract/Free Full Text]
6
6. Grant, C. E., D. L. Sewell, M. Pfaller, R. V. Bumgardner, and J. A. Williams. 1994. Evaluation of two commercial systems for identification of coagulase-negative staphylococci to species level. Diagn. Microbiol. Infect. Dis. 18:1-5.[CrossRef][Medline]
7
7. Heikens, E., A. Fleer, A. Paauw, A. Florijn, and A. C. Flui. 2005. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J. Clin. Microbiol. 43:2286-2290.[Abstract/Free Full Text]
8
8. Ieven, M., J. Verhoeven, S. R. Pattyn, and H. Goossens. 1995. Rapid and economical method for species identification of clinically significant coagulase-negative staphylococci. J. Clin. Microbiol. 33:1060-1063.[Abstract]
9
9. Kloos, W. E., K.-H. Schleifer, and R. Götz. 1991. The genus Staphylococcus, p. 1369-1420. In A. Balows, A. H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.). The procaryotes: a handbook on the biology of bacteria: ecophysiology, isolation, identification, application, 2nd ed. Springer, New York, NY.
10
10. Kontos, F., E. Petinaki, I. Spiliopoulou, M. Maniati, and A. N. Maniatis. 2003. Evaluation of a novel method based on PCR restriction fragment length polymorphism analysis of the tuf gene for the identification of Staphylococcus species. J. Microbiol. Methods 55:465-469.[CrossRef][Medline]
11
11. Kwok, A. Y., and A. W. Chow. 2003. Phylogenetic study of Staphylococcus and Macrococcus species based on partial hsp60 gene sequences. Int. J. Syst. Evol. Microbiol. 53:87-92.[Abstract/Free Full Text]
12
12. Martineau, F., F. J. Picard, D. Ke, S. Paradis, P. H. Roy, M. Ouellette, and M. G. Bergeron. 2001. Development of a PCR assay for identification of staphylococci at genus and species levels. J. Clin. Microbiol. 39:2541-2547.[Abstract/Free Full Text]
13
13. Mellmann, A., K. Becker, C. von Eiff, U. Keckevoet, P. Schumann, and D. Harmsen. 2006. Sequencing and staphylococci identification. Emerg. Infect. Dis. 12:333-336.[Medline]
14
14. Mendoza, M., H. Meugnier, M. Bes, J. Etienne, and J. Freney. 1998. Identification of Staphylococcus species by 16S-23S rDNA intergenic spacer PCR analysis. Int. J. Syst. Bacteriol. 48:1049-1055.[Abstract/Free Full Text]
15
15. Perl, T. M., P. R. Rhomberg, M. J. Bale, P. C. Fuchs, R. N. Jones, F. P. Koontz, and M. A. Pfaller. 1994. Comparison of identification systems for Staphylococcus epidermidis and other coagulase-negative Staphylococcus species. Diagn. Microbiol. Infect. Dis. 18:151-155.[CrossRef][Medline]
16
16. Renneberg, J., K. Rieneck, and E. Gutschik. 1995. Evaluation of Staph ID 32 system and Staph-Zym system for identification of coagulase-negative staphylococci. J. Clin. Microbiol. 33:1150-1153.[Abstract]
17
17. Shah, M. M., H. Iihara, M. Noda, S. X. Song, P. H. Nhung, K. Ohkusu, Y. Kawamura, and T. Ezaki. 2007. dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus. Int. J. Syst. Evol. Microbiol. 57:25-30.[Abstract/Free Full Text]
18
18. Sivadon, V., M. Rottman, J. C. Quincampoix, V. Avettand, S. Chaverot, P. de Mazancourt, P. Trieu-Cuot, and J. L. Gaillard. 2004. Use of sodA sequencing for the identification of clinical isolates of coagulase-negative staphylococci. Clin. Microbiol. Infect. 10:939-942.[CrossRef][Medline]
19
19. Stackebrandt, E., and B. M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:846-849.[Abstract/Free Full Text]
20
20. Stepanovic, S., I. Dakic, D. Morrison, T. Hauschild, P. Jezek, P. Petras, A. Martel, D. Vukovic, A. Shittu, and L. A. Devriese. 2005. Identification and characterization of clinical isolates of members of the Staphylococcus sciuri group. J. Clin. Microbiol. 43:956-958.[Abstract/Free Full Text]
21
21. Sudagidan, M., A. F. Yenidunya, and H. Gunes. 2005. Identification of staphylococci by 16S internal transcribed spacer rRNA gene restriction fragment length polymorphism. J. Med. Microbiol. 54:823-826.[Abstract/Free Full Text]
22
22. Takahashi, T., I. Satoh, and N. Kikuchi. 1999. Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis. Int. J. Syst. Bacteriol. 49:725-728.[Abstract/Free Full Text]
23
23. Vannuffel, P., M. Heusterspreute, M. Bouyer, B. Vandercam, M. Philippe, and J. L. Gala. 1999. Molecular characterization of femA from Staphylococcus hominis and Staphylococcus saprophyticus, and femA-based discrimination of staphylococcal species. Res. Microbiol. 150:129-141.[Medline]
24
24. Yugueros, J., A. Temprano, B. Berzal, M. Sánchez, C. Hernanz, J. M. Luengo, and G. Naharro. 2000. Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp. J. Clin. Microbiol. 38:4351-4355.[Abstract/Free Full Text]
25
25. Yugueros, J., A. Temprano, M. Sánchez, J. M. Luengo, and G. Naharro. 2001. Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism of gap gene. J. Clin. Microbiol. 39:3693-3695.[Abstract/Free Full Text]

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Journal of Clinical Microbiology, December 2008, p. 3875-3879, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00810-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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