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Journal of Clinical Microbiology, December 2001, p. 4296-4301, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4296-4301.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the sodA Gene as a Target

Claire Poyart,^* Gilles Quesne, Claire Boumaila, and Patrick Trieu-Cuot

Laboratoire Mixte Pasteur-Necker de Recherche sur les Streptocoques et Streptococcies and Unité INSERM 411, Faculté de Médecine Necker-Enfants Malades, 75730 Paris Cedex 15, France

Received 20 August 2001/Accepted 28 September 2001

	ABSTRACT

Top Abstract Introduction Materials and Methods Results and Discussion References

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA_int) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Coagulase-negative staphylococci (CNS), which are part of the normal skin flora, have emerged as predominant pathogens in hospital-acquired infections (8, 15). They are associated with the presence of foreign bodies, such as prosthetic valves, cerebrospinal fluid shunts, and orthopedic prostheses, as well as intravascular, urinary, and dialysis catheters. Therefore, it has become increasingly important to accurately identify these isolates to the species level in order to define the clinical significance of these bacteria, to carry out a proper epidemiologic surveillance, and to manage patients infected with CNS in case of relapse. A variety of manual and automated methods have been developed for the identification of CNS that are important in human medicine (6, 7, 19, 24). These methods, based on phenotypic characteristics, include conventional identifications and several commercial kits. Unfortunately, the overall accuracy of these systems is low, ranging from 50 to 70% (6, 7, 19, 24). Several genotypic methods based on the analysis of PCR products derived from selected DNA targets have thus been developed for species-level identification of CNS, including electrophoretic analysis (2) and determination (12) of the 16S ribosomal DNA (rDNA) sequence. In the latter case, however, the interpretation of these data may be complicated by the fact that closely related species may have identical 16S rDNA sequences or, alternatively, that divergent 16S rDNA sequences may exist within a single organism (26). To solve this problem, it is possible to use alternative monocopy target sequences which exhibit a higher divergence than those of the 16S rDNA. Recently, partial sequencing of the highly conserved and ubiquitous hsp60 and tuf genes have been found to be useful for identification and taxonomic classification of species of the genus Staphylococcus (5, 17, 18). It was previously reported that PCR and sequencing of the sodA gene of the gram-positive cocci which encodes the manganese-dependent superoxide dismutase (Mn-SOD), with the use of a single pair of degenerate primers, constitute a valuable approach to the genotypic identification of streptococcal (22) and enterococcal (23) species. In the present study, we report the use of the same universal primers (21) to construct a sodA database of 40 staphylococcal type species and we demonstrate the usefulness of this library for a rapid sequence-based identification method for CNS isolates.

(This study was partially presented at the 100th General Meeting of the American Society for Microbiology, Los Angeles, Calif., 21 to 25 May 2000).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains and culture conditions. The main characteristics of the staphylococcal strains used in this study, including the type strains, are listed in Tables 1 and 2. Type strains were obtained from the Collection de l'Institut Pasteur (CIP). All cultures were grown at 37°C in brain heart infusion broth and subcultured on brain heart infusion agar for examination of the purity and the colony characteristics. Clinical isolates of CNS were identified by the ID 32 Staph system (API-bioMérieux, Marcy l'Etoile, France) according to the manufacturer's instructions and by use of APILAB ID 32 software.

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

View this table: [in this window] [in a new window]   TABLE 2.   Identification of various staphylococcal strains by sequencing the sodAint fragment

DNA manipulations. Rapid extraction of bacterial genomic DNA collected from 2 ml of an overnight culture was performed with the InstaGen Matrix (Bio-Rad) according to the manufacturer's instructions. The sodA degenerate primers d1 (5'-CCITAYICITAYGAYGCIYTIGARCC-3') and d2 (5'-ARRTARTAIGCRTGYTCCCAIACRTC-3') were used to amplify an internal fragment, designated sodA_int, representing approximately 83% of the sodA gene. PCRs were performed on a Gene Amp System 2400 thermal cycler (Perkin-Elmer Cetus, Courtaboeuf, France) in a final volume of 50 µl containing 150 ng of DNA as the template, 0.5 µM each primer, a 200 µM concentration of each deoxynucleoside triphosphate, and 1 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer) in a 1× amplification buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂). The PCR mixtures were denatured (3 min at 95°C) and then subjected to 30 cycles of amplification (60 s of annealing at 37°C, 45 s of elongation at 72°C, and 30 s of denaturation at 95°C). PCR products were resolved by electrophoresis on a 1% agarose gel stained with ethidium bromide. PCR products were purified on an S-400 Sephadex (Pharmacia, Uppsala, Sweden) column, and both strands were directly sequenced with the oligonucleotides d1 and d2 by using an ABI-PRISM Big Dye terminator sequencing kit on an ABI-PRISM 310 Genetic Analyzer (Perkin-Elmer) as previously described (23). All precautions to prevent carryover of amplified DNA were used.

Sequence analysis. Nucleotide sequences were analyzed with Perkin-Elmer software programs (Sequence Analysis, Sequence Navigator, and Autoassembler). Multiple alignment of sod genes was carried out by the CLUSTAL X program (9). The construction of the unrooted phylogenetic tree was performed by the neighbor-joining method (25). The topology of the phylogenetic tree was evaluated by bootstrap analyses to give the degree of confidence intervals for each node on the phylogenetic tree. The confidence values were determined for branches which showed possible monophyletic clades of related organisms separated at each node. It is generally accepted that the monophyly of a clade can be accepted if the clade occurs in more than 95% of the bootstrapped trees (4).

Protein extraction and SOD activity assay. Crude cell lysates of staphylococcal strains were prepared as follows. Cells from 10 ml of overnight cultures were harvested by centrifugation, washed with an equal volume of Tris-EDTA buffer (50 mM Tris HCl [pH 7.6], 50 mM EDTA), suspended in 2 ml of TELL lysis buffer (50 mM Tris HCl [pH 7.6], 50 mM EDTA, 200 mg of lysozyme/liter and 30 mg of lysostaphin/liter), and incubated for 1 h at 37°C. After ultrasonic disruption for 3 min in 30-s pulses at 4°C, the lysates were cleared by centrifugation (16, 170 × g, for 10 min at 4°C). The supernatants were recovered and stored at 20°C until needed. Fifty micrograms of total proteins was electrophoresed through a 10% nondenaturing polyacrylamide gel which was stained for SOD activity by the method of Beauchamp and Fridovich (1).

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

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

CNS express a single Mn-SOD. It has recently been reported that Staphylococcus aureus possesses two genes encoding Mn-SOD, designated sodA and sodM (3, 21, 28). In order to confirm these results, we analyzed the SOD activities of the type strains S. aureus subsp. aureus and S. aureus subsp. anaerobius and 25 unrelated clinical isolates of S. aureus following electrophoresis of protein extracts in a nondenaturing polyacrylamide gel. All strains of S. aureus produce three closely migrating bands of SOD activity (Fig. 1 and data not shown). Previous work demonstrated that the upper band of activity corresponds to SodM, the lowest band corresponds to SodA, and the middle band was proposed to result from the formation of a hybrid protein composed of SodM and SodA (28). This analysis indicates that, unlike all the low-GC-content, gram-positive bacilli and cocci described so far, S. aureus carries genes encoding two unrelated SODs. The fact that S. aureus strains possess two different sod genes impedes direct sequencing of PCR products with the primers d1 and d2, since both genes coamplify with this pair of oligonucleotides. Therefore, to determine if CNS possess one gene encoding Mn-SOD, the SOD activities of crude bacterial extracts of the 40 type strains of CNS were analyzed following electrophoresis in nondenaturing polyacrylamide gels. This analysis revealed that all the strains studied produce a single band of SOD activity (Fig. 1 and data not shown), which suggests that CNS, as opposed to S. aureus, express a single type of Mn-SOD.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   SOD activity gel. Crude extracts (50 µg) of various staphylococcal type strains were loaded onto a nondenaturing 10% polyacrylamide gel stained for SOD activity. Lanes 1 to 18, S. capitis subsp. capitis, S. chromogenes, S. cohnii subsp. cohnii, S. epidermidis, S. haemolyticus, S. hominis subsp. hominis, S. hyicus; S. intermedius, S. lentus, S. lugdunensis, S. saprophyticus subsp. saprophyticus, S. schleiferi subsp. schleiferi, S. simulans, S. vitulus, S. warneri, S. xylosus, S. aureus subsp. aureus, and S. aureus subsp. anaerobius, respectively.

Amplification and sequencing of the sodA_int gene from various CNS type strains. By using the primers d1 and d2 in a PCR assay, we amplified an internal fragment representing approximately 85% of the sodA gene in 40 type strains of CNS (Staphylococcus arlettae, Staphylococcus auricularis, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus caprae, Staphylococcus carnosus subsp. carnosus, Staphylococcus carnosus subsp. utilis, Staphylococcus chromogenes, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus condimenti, Staphylococcus delphini, Staphylococcus epidermidis, Staphylococcus equorum, Staphylococcus felis, Staphylococcus gallinarum, Staphylococcus haemolyticus, Staphylococcus hominis subsp. hominis, Staphylococcus hominis subsp. novobiosepticus, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus lutrae, Staphylococcus muscae, Staphylococcus pasteuri, Staphylococcus piscifermentans, Staphylococcus pulvereri, Staphylococcus saccharolyticus, Staphylococcus saprophyticus subsp. bovis, Staphylococcus saprophyticus subsp. saprophyticus, Staphylococcus schleiferi subsp. coagulans, Staphylococcus schleiferi subsp. schleiferi, Staphylococcus sciuri subsp. carnaticus, Staphylococcus sciuri subsp. sciuri, Staphylococcus simulans, Staphylococcus vitulus, Staphylococcus warneri, Staphylococcus xylosus). We also included in this study Macrococcus caseolyticus, which was formerly designated Staphylococcus caseolyticus (13). A single amplification product having the expected size of 480 bp was observed with all staphylococcal species (data not shown). Direct sequencing of these amplicons gave rise to electropherograms devoid of overlapping peaks, which confirms that these strains contain a single type of sod gene. Sequence analysis of these amplicons revealed that they were actual sodA_int fragments, since the corresponding deduced polypeptides all possessed the amino acids characteristic of the Mn-SOD at the expected positions (data not shown). Multiple alignment of the staphylococcal sodA_int DNA sequences was carried out by the CLUSTAL X program. The sequences of the degenerated primers d1 and d2 and alignment gaps were not taken into consideration for calculations. Although some differences could be observed, the topology of the phylogenetic tree obtained (Fig. 2) was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences (17, 27). The phylogenetic position of M. caseolyticus is the most distant from any other CNS species (Fig. 2 and data not shown), an observation consistent with the decision to remove this species from the genus Staphylococcus (13). It is worth noting, however, that most staphylococcal species groups are not supported by significant bootstrap values (i.e., 95%). In fact, if this critical value is used, only three major clusters corresponding to species groups S. sciuri, S. intermedius, and S. simulans are defined (Fig. 2).

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Phylogenetic unrooted tree showing relationships among the sodAint fragments from various staphylococcal type strains. The tree was established from an analysis of the sequences listed in Table 1 by using the neighbor-joining method. The sodAint sequence of M. caseolyticus type strains included in this work was used as an outgroup sequence to root the tree. The value on each branch is the estimated confidence limit (expressed as a percentage) for the position of the branch as determined by bootstrap analysis. Only the bootstrap values superior to 95% were considered significant (4). The scale bar (neighbor-joining distance) represents 10% differences in nucleotide sequences. The accession numbers of sodA and sodM were AF121672 and Z49245, respectively.

The S. sciuri group includes S. sciuri, S. lentus, S. pulvereri, and S. vitulus. These four species differ from the other Staphylococcus species by several remarkable features. They, are novobiocin resistant and oxidase positive, they are the sole species possessing cytochrome c in their electron transport systems, and they all share the same characteristic pattern of amino acid substitution in their HSP60 proteins (14, 17). Interestingly, we observed that their sodA_int sequences, when compared to other CNS sequences, contained an additional codon which codes for a prolyl residue at position 78 of the corresponding 143-amino-acid-long partial SodA protein (data not shown). It is notable that the sodA_int sequences of S. pulvereri and S. vitulus display 99.5% identity, which confirms that they constitute a single species (20).

An S. intermedius group consisting of S. intermedius, S. delphini, and S. lutrae was recovered in 98.1% of the bootstrap trees. The fact that S. schleiferi was not included in this cluster is in disagreement with the results from DNA annealing studies or from a phylogenetic analysis of their 16S rDNA or hsp60 gene sequences (17, 27). Moreover, the related species S. hyicus, S. muscae, and S. chromogenes did not cluster to form a S. hyicus species subgroup (Fig. 2).

The S. simulans group species, defined in 100% of the bootstrap trees (Fig. 2), consists of S. simulans, S. piscifermentans, S. condimenti, and S. carnosus.

The S. saprophyticus group, as defined by 16S rDNA sequence analysis (91% of bootstrap value), includes the novobiocin-resistant and oxidase-negative species S. saprophyticus, S. arlettae, S. kloosi, S. cohnii, S. gallinarum, S. equorum, and S. xylosus (27). In our analysis, however, the monophyly of this clade is uncertain since it is associated with a bootstrap value of 68% (Fig. 2). Similarly, the S. epidermidis group (S. epidermidis, S. capitis, S. caprae, and S. saccharolyticus), which constitutes a monophyletic clade supported by a high bootstrap value (97%) on the basis of 16S rDNA sequence analysis (27), did not form a clearly distinct lineage in our study (38.9% of bootstrap value). On the other hand, association of S. pasteuri and S. warneri to the S. epidermidis group was inferred from our treeing analysis (Fig. 2).

As reported in an analysis of their 16S rDNA sequences (27), we found that the branching order of the species S. auricularis, S. haemolyticus, S. hominis, and S. lugdunensis was uncertain in our sodA_int-based phylogenetic analysis.

Evidence for horizontal transfer of a sod gene from CNS to S. aureus. The sodA and sodM genes from S. aureus display 77.5% sequence identity, which indicates that the presence of these two isofunctional genes in this bacterium is not due to a recent duplication event. In a sodA_int phylogenetic tree that includes the sequences of Bacillus subtilis, Clostridium perfringens, Enterococcus faecalis, Enterococus faecium, Lactococcus lactis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, both the sodM and sodA genes from S. aureus were clearly positioned within the staphylococcal lineage (data not shown). However, it is worth noting that the phylogenetic position of the sodM gene in the sod tree (Fig. 2) is similar to that of S. aureus in 16S rDNA or hsp60 gene trees (17, 27). This might indicate that sodM is the indigenous S. aureus sod gene whereas sodA was acquired by horizontal gene transfer from an as-yet-uncharacterized CNS. This hypothesis is based on the fact that S. aureus possesses a remarkable ability to acquire useful genes from various bacteria by lateral gene transfer, as revealed by genome sequence analysis (16). Accordingly, it has been proposed that the mecA homologue present in S. sciuri is the evolutionary precursor of the S. aureus methicillin resistance gene mecA (29). Efforts are currently being made to track the original host of the S. aureus sodA gene.

Species identification of clinical and environmental isolates of staphylococci by sequencing the sodA_int gene. Pairwise comparison of the staphylococcal sodA_int sequences revealed that their mean identity (81.5%) is inferior to that calculated from a comparison of their 16S rDNA genes (mean identity, 98%) but is similar to that computed from a comparison of their hsp60 genes (mean identity, 82%) (17). These results confirm that sodA might constitute a more discriminative target sequence than does the 16S RNA to differentiate closely related bacterial species, as already demonstrated for differentiating closely related species belonging to the genera Streptococcus and Enterococcus (11, 22, 23). The sodA_int fragments of S. cohnii subsp. cohnii and S. cohnii subsp. urealyticum display 4% sequence divergence, enabling the distinction between these two subspecies (Table 3). However, the sodA_int fragments of the remaining pairs of type strain subspecies (S. capitis subsp. capitis and S. capitis subsp. ureolyticus, S. carnosus subsp. carnosus and S. carnosus subsp. utilis, S. hominis subsp. hominis and S. hominis subsp. novobiosepticus, S. saprophyticus subsp. bovis and S. saprophyticus subsp. saprophyticus, S. schleiferi subsp. coagulans and S. schleiferi subsp. schleiferi, S. sciuri subsp. carnaticus and S. sciuri subsp. sciuri) display more than 99.3% sequence identity. This finding is consistent with the observation that the hsp60 genes of S. schleiferi subsp. coagulans and S. schleiferi subsp. schleiferi display 98% sequence identity (17). We therefore concluded that the sodA gene, like the hsp60 gene, does not allow discrimination at the subspecies level.

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

Twenty-six unrelated CNS isolates were identified by conventional microbiological tests, the ID 32 Staph system, and the sodA_int system (Table 2). In all cases, the sodA_int sequences of the isolates displayed less than 1.5% divergence with that of the corresponding type strain (Table 2). For 14 strains (NEM1999, NEM2000, NEM2002, NEM2004, NEM2006, NEM2008, NEM2009, NEM2010, NEM2011, NEM2013, NEM2014, NEM2016, NEM2021, and NEM2022), the two methods gave the same results. The remaining 12 isolates were not identified at the species level, or were misidentified, by the conventional microbiological phenotypic tests. These strains were correctly identified by the sodA_int system. For example, NEM1997 and NEM1998 were identified by the ID 32 Staph system as S. capitis although they were novobiocin resistant and oxidase positive. Both strains were identified as M. caseolyticus following sodA_int-based sequence analysis. NEM2001, NEM2003, NEM2004, NEM2015, and NEM2018 were formerly misidentified by conventional methods as S. hominis, S. epidermidis, S. haemolyticus, S. warneri, and S. epidermidis, respectively. They were subsequently unambiguously identified with the sodA system as S. capitis, S. caprae, S. pasteuri, and S. simulans, respectively. These latter species are often misidentified by conventional methods (10), and genotypic methods are often necessary to identify these uncommon species. Lastly, NEM2005, NEM2012, NEM2017, NEM2019, and NEM2020, which were not identified by the ID 32 Staph system, were identified by the sodA_int method as S. caprae, S. haemolyticus, S. schleiferi, S. vitulus, and S. warneri, respectively.

Conclusions. We have determined the sodA_int sequences of 40 type strains of CNS and demonstrated the usefulness of this database for species-level identification of staphylococcal isolates. This method consists of a PCR carried out with a single pair of degenerate oligonucleotides for amplification of a staphylococcal sodA_int fragment and direct sequencing of the PCR product with the same degenerate primers. Under these conditions, the delay required for bacterial identification is less than 24 h. This method might be useful in reference laboratories for characterization of strains that could not be assigned to a species on the basis of their conventional phenotypic reaction. Furthermore, the sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.

	ACKNOWLEDGMENTS

We thank C. Bizet for the gift of staphylococcal type strains (CIP) and S. Nair for critical reading of the manuscript.

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

	FOOTNOTES

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

	REFERENCES

Top Abstract Introduction Materials and Methods Results and Discussion References

1.	Beauchamp, C., and I. Fridovich. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44:276-287[CrossRef][Medline].
2.	Chesneau, O., A. Morvan, S. Aubert, and N. el Solh. 2000. The value of rRNA gene restriction site polymorphism analysis for delineating taxa in the genus Staphylococcus. Int. J. Syst. Evol. Microbiol. 50:689-697[Abstract].
3.	Clements, M. O., S. P. Watson, and S. J. Foster. 1999. Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity. J. Bacteriol. 181:3898-3903[Abstract/Free Full Text].
4.	Felsenstein, J. 1985. Confidence limits on phylogeny and approach using the boostrap. Evolution 39:783-791[CrossRef].
5.	Goh, S. H., S. Potter, J. O. Wood, S. M. Hemmingsen, R. P. Reynolds, and A. W. Chow. 1996. HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J. Clin. Microbiol. 34:818-823[Abstract].
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.	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].
8.	Jarvis, W. R., and W. J. Martone. 1992. Predominant pathogens in hospital infections. J. Antimicrob. Chemother. 29:19-24[Abstract/Free Full Text].
9.	Jeanmougin, F., J. D. Thompson, M. Gouy, D. G. Higgins, and T. J. Gibson. 1998. Multiple sequence alignment with Clustal X. Trends Biochem. Sci. 23:403-405[CrossRef][Medline].
10.	Kawamura, Y., X.-G. Hou, F. Sultana, K. Hirose, M. Miyake, S.-E. Shu, and T. Ezaki. 1998. Distribution of Staphylococcus species among human clinical specimens and emended description of Staphylococcus caprae. J. Clin. Microbiol. 36:2038-2042[Abstract/Free Full Text].
11.	Kawamura, Y., R. A. Whiley, S. E. Shu, T. Ezaki, and J. M. Hardie. 1999. Genetic approaches to the identification of the mitis group within the genus Streptococcus. Microbiology 145:2605-2613[Abstract/Free Full Text].
12.	Kloos, W. E. 1997. Taxonomy and systematics of staphylococci indigenous to humans, p. 113-137. In K. B. Croosley, and G. L. Archer (ed.), The staphylococci in human disease. Churchill Livingston, New York, N.Y.
13.	Kloos, W. E., D. N. Ballard, C. G. George, J. A. Webster, R. J. Hubner, W. Ludwig, K. H. Schleifer, and K. Schubert. 1998. Delimiting the genus Staphylococcus through description of Macrococcus caseolyticus gen. nov., comb. nov. and Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. and Macrococcus carouselicus sp. nov. Int. J. Syst. Bacteriol. 48:859-877[Abstract/Free Full Text].
14.	Kloos, W. E., and T. L. Bannerman. 1995. Staphylococcus and Micrococcus, p. 282-298. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
15.	Kloos, W. E., and T. L. Bannerman. 1994. Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev. 7:117-140[Abstract/Free Full Text].
16.	Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357:1225-1240[CrossRef][Medline].
17.	Kwok, A. Y., S. C. Su, R. P. Reynolds, S. J. Bay, Y. Av-Gay, N. J. Dovichi, and A. W. Chow. 1999. Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus. Int. J. Syst. Bacteriol. 49:1181-1192[Abstract/Free Full Text].
18.	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].
19.	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].
20.	Petras, P. 1998. Staphylococcus pulvereri = Staphylococcus vitulus? Int. J. Syst. Bacteriol. 48:617-618[Free Full Text].
21.	Poyart, C., P. Berche, and P. Trieu-Cuot. 1995. Characterization of superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers. FEMS Microbiol. Lett. 131:41-45[CrossRef][Medline].
22.	Poyart, 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-dependent superoxide dismutase. J. Clin. Microbiol. 36:41-47[Abstract/Free Full Text].
23.	Poyart, C., G. Quesnes, and P. Trieu-Cuot. 2000. Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci. J. Clin. Microbiol. 38:415-418[Abstract/Free Full Text].
24.	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].
25.	Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425[Abstract].
26.	Stackebrandt, E., and B. M. Goebel. 1994. 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].
27.	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].
28.	Wright Valderas, M., and M. E. Hart. 2001. Identification and characterization of a second superoxide dismutase gene (sodM) from Staphylococcus aureus. J. Bacteriol. 183:3399-3407[Abstract/Free Full Text].
29.	Wu, S. W., H. de Lencastre, and A. Tomasz. 2001. Recruitment of the mecA gene homologue of Staphylococcus sciuri into a resistance determinant and expression of the resistant phenotype in Staphylococcus aureus. J. Bacteriol. 183:2417-2424[Abstract/Free Full Text].

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Journal of Clinical Microbiology, December 2001, p. 4296-4301, Vol. 39, No. 12
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.12.4296-4301.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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