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Journal of Clinical Microbiology, October 2007, p. 3302-3308, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01082-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Comparison of Single- and Multilocus Sequence Typing and Toxin Gene Profiling for Characterization of Methicillin-Resistant Staphylococcus aureus{triangledown}

Yongwei Cai,1,2 Fanrong Kong,1 Qinning Wang,1 Zhongsheng Tong,1,3 Vitali Sintchenko,1 Xianyu Zeng,3 and Gwendolyn L. Gilbert1*

Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia,1 Department of Dermatology, Hangzhou Third People's Hospital, Hangzhou, Zhejiang Province, People's Republic of China,2 Research Laboratory for Infectious Skin Diseases, Department of Dermatology, Wuhan First Hospital, Wuhan 430022, People's Republic of China3

Received 28 May 2007/ Returned for modification 2 August 2007/ Accepted 15 August 2007


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ABSTRACT
 
We compared three novel methicillin-resistant Staphylococcus aureus (MRSA) genotyping methods with multilocus sequence typing (MLST) and spa typing to assess their utility for routine strain typing. The new methods were femA and nuc sequence typing and toxin gene profiling (TGP), using a multiplex-PCR-based reverse line blot assay to detect 13 pyrogenic superantigen and exfoliative toxin genes. Forty-two well-characterized MRSA strains, representing 15 MLSTs or 9 clonal clusters (CCs), were genotyped by all methods. Twenty-two spa, nine femA, and seven nuc sequence types were identified. The femA sequence types correlated exactly with CCs; nuc sequences types were less discriminatory but generally correlated well with femA types and CCs. Ten isolates contained none of 13 toxin genes; TGPs of the remainder comprised 1 to 5 toxin genes. The combination of spa typing and TGPs identified 26 genotypes among the 42 strains studied. A combination of two or three rapid, inexpensive genotyping methods could potentially provide rapid MRSA strain typing as well as useful information about clonal origin and virulence.


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INTRODUCTION
 
Methicillin-resistant Staphylococcus aureus (MRSA) genotyping is used to study its evolution and epidemiology and to assist in infection control (39). Different typing methods provide different information. Multilocus sequence typing (MLST) reveals slowly accumulating changes in conserved genes that reflect long-term evolutionary changes and can identify global spread of the relatively small number of successful clones (13). It has limited discriminatory power and is unsuitable for outbreak investigation, whereas pulsed-field gel electrophoresis is highly discriminatory and can identify recent changes. It is most widely used for outbreak investigation and infection control (9, 28). Both methods are relatively expensive and slow, and a number of rapid, inexpensive typing methods, based on sequence or length polymorphisms of variable genes or loci, have been described that are objective and relatively inexpensive. These include coa (41) and spa (38) sequence typing and the multilocus variable number tandem repeat assay (12, 29).

spa typing, which depends on differences in the number and sequence of tandem repeats in region X of the protein A gene (44), is discriminatory, rapid, inexpensive, and objective (25, 37, 41). The development of a shareable web-based database (www.spaServer.Ridom.de) (15) and the utility of spa typing for early-warning systems (31) have contributed to the rapid uptake of MRSA spa typing by diagnostic and public health laboratories.

In this study, we investigated the potential utility of two additional S. aureus gene polymorphisms for strain typing, namely, femA, one of several genes involved in the synthesis of the branched-peptide structure of S. aureus peptidoglycan (4), and nuc, which encodes an extracellular thermostable nuclease of S. aureus (5). Both are species-specific S. aureus genes; they have been widely used as PCR targets for identification (21), but their polymorphisms have not been widely investigated (14).

S. aureus produces numerous toxins, including enterotoxins or pyrogenic superantigens and exfoliative toxins, some of which are encoded by genes carried on staphylococcal pathogenicity islands and associated with certain clonal complexes (CCs), whereas genes encoding others, such as the Panton-Valentine leucocidin (PVL), are carried on bacteriophages and readily transferred between different lineages (26, 27). This suggests that a toxin gene profile (TGP) could help identify S. aureus CCs as well as providing information about virulence. Various molecular methods have been described for studying the distribution of staphylococcal toxins (2, 10).

We used 42 well-characterized MRSA strains to compare sequence polymorphisms of femA and nuc and TGPs, based on a multiplex PCR-based reverse line blot assay (mPCR/RLB) (22), with two established typing methods—namely, spa typing and MLST—to determine their potential utility for MRSA genotyping.


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MATERIALS AND METHODS
 
S. aureus isolates. We used 42 well-characterized reference and clinical S. aureus isolates in this study, as shown in Table 1, including 35 from various parts of Australia, provided by Philip Giffard, Cooperative Research Centre for Diagnostics, Queensland University of Technology, Brisbane, and Graeme Nimmo, Queensland Health Pathology Services, Princess Alexandra Hospital, Brisbane, Australia. Some have been used in several previous studies (40). Seven strains were provided by Herminia de Lencastre, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal, and also have been used in previous studies (33, 34) Two of these strains (MW2 and COL) have been fully sequenced (26). MLST and SCCmec typing results were provided by the donors of the strains (33, 34, 40).


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  		TABLE 1. Genotypes and spa types of 42 well-characterized methicillin-resistant S. aureus isolates used in this study

DNA extraction. DNA extraction was performed as described previously (23).

Toxin gene detection. A well-established mPCR/RLB protocol developed in our laboratory (22) was used to detect 13 Staphylococcus aureus toxin genes. Target genes, primer and probe sequences, physical characteristics, and locations within selected GenBank sequences are shown in Table 2. All primers and probes had similar physical characteristics to allow simultaneous amplification and hybridization, respectively, in a multiplex reaction (22). Two gene-specific PCR primer pairs and two gene-specific probes were designed for each of 13 toxin genes. All primers were 5' end biotinylated to allow detection of hybridization with a streptavidin peroxidase substrate. The probes were labeled with a 5'-end amine group to facilitate covalent linkage to the nylon membrane and to allow membranes to be stripped and reused repeatedly (22). Each multiplex reaction included nuc primers as the positive control for S. aureus and for quality control of DNA extraction and mPCR/RLB. All primers and probes were synthesized by Sigma-Aldrich (Sydney, Australia).


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  		TABLE 2. Primers and probes used in mPCR/RLB for detection of 13 toxin genes

The mPCR/RLB was performed as previously described (22) with the following modifications: each 25-µl reaction mixture contained 0.5 U Hotstar Taq polymerase (QIAGEN, Melbourne, Australia), and the mPCR annealing temperature was optimized to 55°C.

Sequencing, sequence analysis, and phylogenetic tree. femA, nuc, and spa PCR primers were based on the published GenBank sequences using BioManager (http://biomanager.angis.org.au/). Sequencing was performed as described previously (24). For most targets, outer primers were used for amplification and inner primers for sequencing (Table 3).


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  		TABLE 3. Primers used for PCR sequencing of nuc, femA, and spa genes

The spa types were defined by reference to the shareable web-based database (www.spaServer.Ridom.de) (15). All spa repeat regions were submitted to the database, and spa types were assigned by the database by combining the sequences of all repeat regions.

Data obtained from different typing methods were recorded and stored in an Access file, which was imported into the BioNumerics software program (Applied Maths) with appropriate formatting. A phylogenetic tree was generated by using the categorical coefficient and clustered by the Ward algorithm.

Calculation of index of diversity. Simpson's index of diversity was calculated for each individual genotyping method and for combinations of methods, as described by Hunter and Gaston (16).

Nucleotide sequence accession numbers. The nearly full-length sequences (see below) of selected femA and nuc genes and partial spa sequences were deposited in GenBank with the following accession numbers: for femA, DQ103589 and DQ352456 to DQ352463; for nuc, DQ507377 to DQ507382; and for spa, EF094507 to EF094528. Eight S. aureus genome sequences were used for reference: AJ938182 (RF122), NC_002952 (MRSA252), AF144661 (Staphylococcus aureus subsp. anaerobius), NC_002745(N315), NC_002758 (Mu50), NC_003923 (MW2), NC_002951 (COL), and NC_002953 (MSSA476).


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RESULTS AND DISCUSSION
 
Sequence polymorphisms of femA and nuc. Nine femA sequence types with lengths of approximately 1,215 bp were identified, of which five were newly identified and four had been previously deposited in GenBank. A total of 39 polymorphism sites were found among those sequences. Seven nuc sequence types of approximately 700 bp were identified, with 28 polymorphisms sites. Four sequence types had not been previously identified.

spa types and TGPs. Twenty-two spa types were identified, and sequences of each new type were submitted to GenBank with accession numbers shown in Table 1. Nineteen of the spa types were already recorded in the spa database (www.spaServer.Ridom.de) (15), and three were new types, first identified in Australian strains (Table 1; Fig. 1). There were 14 different TGPs (Fig. 1 and 2).


Figure 1
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  		FIG. 1. Relatedness of 42 MRSA strains between different typing methods. *, strains CH 69, IPO1M2046, and 14176-5710 belonged to spa types t1963, t1958, and t1959, respectively, which have not been previously deposited in the database.


Figure 2
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  		FIG. 2. The 13 toxin gene profiles of the 41 strains. Lanes 1 to 42 show results for the following isolates, in order (see Table 1): FH43, SJOG 30, RPH85, B827549, SN39, RHH58, RHH10, FH53, B8-10, RPH2, IMVS67, CH16, CH69, PAH58, PAH1, E822485, J710566, RPH74, E804531, CH97, IP01M2046, C801535, F829549, RBH98, 13792-4492, IP01M1081, 14176-5710, K704540, K711532, AH13, RDH81, AH1, RPAH18, RPAH15, COL, MW2, DEN2988, BK2464, HDG2, HU25, a control strain, and ANS46.

Comparison of MLST, femA and nuc sequence types, spa types, and TGPs. MLST are based on sequences of seven housekeeping genes (http://www.mlst.net/). Isolates with identical sequences for all seven genes are considered to be clonal and those with five or six matching genes to belong to the same CC (27). There were 15 MLST and nine CCs among the 42 strains studied (Table 1). The latter correlated exactly with femA sequence types, suggesting that femA sequencing may be a useful "shorthand" single-locus surrogate for MLST (Fig. 1). In future, informative single nucleotide polymorphisms in the femA sequence may be able to predict femA type and CC. One of the candidate methods is rolling-circle amplification, which has been used successfully in our laboratory (42).

There were seven nuc sequence types, which were therefore less informative. One sequence type was represented among three femA sequence types (and corresponding CCs).

The relationships between MLST and TGP varied (Fig. 1). No toxin genes were found in 10 isolates belonging to four sequence types (STs) (ST-8, -78, -88, and -239). One to five toxin genes were found in various combinations in the remaining isolates. Some STs included more than one TGP, e.g., ST-5, -22, -45, and -239 each included isolates with two different TGPs, which reflects the ability of mobile genetic elements on which toxin genes are carried to transfer laterally between clones.

However, some toxin genes are transferred vertically within specific CCs (32, 36). For example, all 10 ST-1 isolates (but none belonging to other STs) contained sea and seh; sea alone was found in another eight isolates belonging to CC 8/239. seg and sei, which are part of the enterotoxin gene complex (egc), were always present together, in 10 isolates spread among four CCs (ST-5/73, -22, -45, and -30/36). This is consistent with a previous report that egc is preferentially distributed among CCs 5 and 30/36 (19). In addition, we identified mutations in regions of seg and sei probes in isolates belonging to CC 30/36.

The Panton-Valentine leukocidin gene (pvl) was identified, with a variety of other toxin genes (depending on ST/CC), in eight isolates belonging to ST-1 (three isolates), ST-93 (two isolates), or CC 30/36 (three isolates); seven of eight PVL-containing isolates belonged to SCCmec type IV, which is generally associated with community-acquired MRSA. PVL is associated with necrotic skin and soft tissue lesions and, more recently, with life-threatening necrotizing pneumonia and sepsis due to community-acquired MRSA (8, 44).

There were 22 spa types among the 42 strains tested. When combined with TGP, some spa types were further subdivided, making a total of 26 genotypes. For example, isolates belonging to spa type t002 contained two TGPs (seg-sei and sed-seg-sei), and those belonging to t008 contained three (sea, seb, and none). The combination of these two methods thus provides a high level of discrimination, using relatively inexpensive, rapid methods.

Comparison of discriminatory powers of each genotyping method and various combinations (Table 4) showed that spa typing is the most discriminatory. The addition of TGPs alone or TGP plus SCCmec typing increases the discriminatory power, but there is little additional increase from additional femA or nuc sequence typing.


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  		TABLE 4. Comparison of discriminatory powers of each genotyping method and various combinations of methods for 42 MRSA strains using Simpson index of diversity

Significance of sequence polymorphisms. These results indicate that femA and to some extent nuc sequence types correlate closely with MLST in this set of MRSA isolates, suggesting that these genes evolve at a rate similar to that of housekeeping genes within CCs (20, 27). spa types were more discriminatory for strain typing but correlated less well with MLST or CCs.

Significant sequence variation in femA and nuc also has potential implications for their use as species-specific PCR targets for identification of S. aureus. The possibility of mutations needs to be considered in the design of probes and primers to avoid false-negative or inaccurate quantitative PCR results. Our results show that some mutations occurred in the region of primers used as species-specific primers (1).

There were significant sequence polymorphisms in femA and nuc genes, which have not been previously well studied, which has potential implications for their use as species-specific PCR targets for identification of S. aureus. Both correlated well with each other, and femA sequence types correlated with MLST/CCs. TGPs provide useful information about potential virulence and the evolutionary history of S. aureus strains and can increase the discriminatory power of femA and spa sequence typing. Prospective testing of unselected clinical isolates will be needed to adequately determine the optimal combination of methods for MRSA surveillance.


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ACKNOWLEDGMENTS
 
We sincerely thank the following colleagues for allowing us to study their isolates: Herminia de Lencastre, Philip Giffard, and Graeme Nimmo.

Fanrong Kong, Qinning Wang, and Yongwei Cai made similar contributions to this work and so would be seen as co-first authors.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyng{at}icpmr.wsahs.nsw.gov.au Back

{triangledown} Published ahead of print on 22 August 2007. Back


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REFERENCES
 
    1
  1. Alarcon, B., B. Vicedo, and R. Aznar. 2006. PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. J. Appl. Microbiol. 100:352-364.[CrossRef][Medline]
    2. 2
  2. Becker, K., A. W. Friedrich, G. Lubritz, M. Weilert, G. Peters, and C. von Eiff. 2003. Prevalence of genes encoding pyrogenic toxin superantigens and exfoliative toxins among strains of Staphylococcus aureus isolated from blood and nasal specimens. J. Clin. Microbiol. 41:1434-1439.[Abstract/Free Full Text]
    3. 3
  3. Becker, K., R. Roth, and G. Peters. 1998. Rapid and specific detection of toxigenic Staphylococcus aureus: use of two multiplex PCR enzyme immunoassays for amplification and hybridization of staphylococcal enterotoxin genes, exfoliative toxin genes, and toxic shock syndrome toxin 1 gene. J. Clin. Microbiol. 36:2548-2553.[Abstract/Free Full Text]
    4. 4
  4. Berger-Bachi, B., and S. Rohrer. 2002. Factors influencing methicillin resistance in staphylococci. Arch. Microbiol. 178:165-171.[CrossRef][Medline]
    5. 5
  5. Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654-1660.[Abstract/Free Full Text]
    6. 6
  6. Coombs, G. W., G. R. Nimmo, J. M. Bell, F. Huygens, F. G. O'Brien, M. J. Malkowski, J. C. Pearson, A. J. Stephens, and P. M. Giffard. 2004. Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia. J. Clin. Microbiol. 42:4735-4743.[Abstract/Free Full Text]
    7. 7
  7. Costa, A. M., I. Kay, and S. Palladino. 2005. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 51:13-17.[CrossRef][Medline]
    8. 8
  8. Denis, O., A. Deplano, H. De Beenhouwer, M. Hallin, G. Huysmans, M. G. Garrino, Y. Glupczynski, X. Malaviolle, A. Vergison, and M. J. Struelens. 2005. Polyclonal emergence and importation of community-acquired methicillin-resistant Staphylococcus aureus strains harbouring Panton-Valentine leucocidin genes in Belgium. J. Antimicrob. Chemother. 56:1103-1106.[Abstract/Free Full Text]
    9. 9
  9. Deplano, A., R. De Mendonca, R. De Ryck, and M. J. Struelens. 2006. External quality assessment of molecular typing of Staphylococcus aureus isolates by a network of laboratories. J. Clin. Microbiol. 44:3236-3244.[Abstract/Free Full Text]
    10. 10
  10. Ferry, T., D. Thomas, A. L. Genestier, M. Bes, G. Lina, F. Vandenesch, and J. Etienne. 2005. Comparative prevalence of superantigen genes in Staphylococcus aureus isolates causing sepsis with and without septic shock. Clin. Infect. Dis. 41:771-777.[CrossRef][Medline]
    11. 11
  11. Francois, P., G. Renzi, D. Pittet, M. Bento, D. Lew, S. Harbarth, P. Vaudaux, and J. Schrenzel. 2004. A novel multiplex real-time PCR assay for rapid typing of major staphylococcal cassette chromosome mec elements. J. Clin. Microbiol. 42:3309-3312.[Abstract/Free Full Text]
    12. 12
  12. Gilbert, F. B., A. Fromageau, L. Gelineau, and B. Poutrel. 2006. Differentiation of bovine Staphylococcus aureus isolates by use of polymorphic tandem repeat typing. Vet. Microbiol. 117:297-303.[CrossRef][Medline]
    13. 13
  13. Gomes, A. R., S. Vinga, M. Zavolan, and H. de Lencastre. 2005. Analysis of the genetic variability of virulence-related loci in epidemic clones of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 49:366-379.[Abstract/Free Full Text]
    14. 14
  14. Hamels, S., J. L. Gala, S. Dufour, P. Vannuffel, N. Zammatteo, and J. Remacle. 2001. Consensus PCR and microarray for diagnosis of the genus Staphylococcus, species, and methicillin resistance. BioTechniques 31:1364-1366, 1368, 1370-1372.[Medline]
    15. 15
  15. Harmsen, D., H. Claus, W. Witte, J. Rothganger, H. Claus, D. Turnwald, and U. Vogel. 2003. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J. Clin. Microbiol. 41:5442-5448.[Abstract/Free Full Text]
    16. 16
  16. Hunter, P. R., and M. A. Gaston. 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26:2465-2466.[Abstract/Free Full Text]
    17. 17
  17. Huygens, F., J. Inman-Bamber, G. R. Nimmo, W. Munckhof, J. Schooneveldt, B. Harrison, J. A. McMahon, and P. M. Giffard. 2006. Staphylococcus aureus genotyping using novel real-time PCR formats. J. Clin. Microbiol. 44:3712-3719.[Abstract/Free Full Text]
    18. 18
  18. Huygens, F., A. J. Stephens, G. R. Nimmo, and P. M. Giffard. 2004. mecA locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone. J. Clin. Microbiol. 42:1947-1955.[Abstract/Free Full Text]
    19. 19
  19. Jarraud, S., G. Cozon, F. Vandenesch, M. Bes, J. Etienne, and G. Lina. 1999. Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever. J. Clin. Microbiol. 37:2446-2449.[Abstract/Free Full Text]
    20. 20
  20. Jarraud, S., C. Mougel, J. Thioulouse, G. Lina, H. Meugnier, F. Forey, X. Nesme, J. Etienne, and F. Vandenesch. 2002. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70:631-641.[Abstract/Free Full Text]
    21. 21
  21. Kizaki, M., Y. Kobayashi, and Y. Ikeda. 1994. Rapid and sensitive detection of the femA gene in staphylococci by enzymatic detection of polymerase chain reaction (ED-PCR): comparison with standard PCR analysis. J. Hosp. Infect. 28:287-295.[CrossRef][Medline]
    22. 22
  22. Kong, F., and G. L. Gilbert. 2006. Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool. Nat. Protoc. 1:2668-2680.[CrossRef][Medline]
    23. 23
  23. Kong, F., S. Gowan, D. Martin, G. James, and G. L. Gilbert. 2002. Molecular profiles of group B streptococcal surface protein antigen genes: relationship to molecular serotypes. J. Clin. Microbiol. 40:620-626.[Abstract/Free Full Text]
    24. 24
  24. Kong, F., S. Gowan, D. Martin, G. James, and G. L. Gilbert. 2002. Serotype identification of group B streptococci by PCR and sequencing. J. Clin. Microbiol. 40:216-226.[Abstract/Free Full Text]
    25. 25
  25. Koreen, L., S. V. Ramaswamy, E. A. Graviss, S. Naidich, J. M. Musser, and B. N. Kreiswirth. 2004. spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation. J. Clin. Microbiol. 42:792-799.[Abstract/Free Full Text]
    26. 26
  26. Lindsay, J. A., and M. T. Holden. 2004. Staphylococcus aureus: superbug, super genome? Trends Microbiol. 12:378-385.[CrossRef][Medline]
    27. 27
  27. Lindsay, J. A., C. E. Moore, N. P. Day, S. J. Peacock, A. A. Witney, R. A. Stabler, S. E. Husain, P. D. Butcher, and J. Hinds. 2006. Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J. Bacteriol. 188:669-676.[Abstract/Free Full Text]
    28. 28
  28. Macfarlane, L., J. Walker, R. Borrow, B. A. Oppenheim, and A. J. Fox. 1999. Improved recognition of MRSA case clusters by the application of molecular subtyping using pulsed-field gel electrophoresis. J. Hosp. Infect. 41:29-37.[CrossRef][Medline]
    29. 29
  29. Malachowa, N., A. Sabat, M. Gniadkowski, J. Krzyszton-Russjan, J. Empel, J. Miedzobrodzki, K. Kosowska-Shick, P. C. Appelbaum, and W. Hryniewicz. 2005. Comparison of multiple-locus variable-number tandem-repeat analysis with pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing for clonal characterization of Staphylococcus aureus isolates. J. Clin. Microbiol. 43:3095-3100.[Abstract/Free Full Text]
    30. 30
  30. Mehrotra, M., G. Wang, and W. M. Johnson. 2000. Multiplex PCR for detection of genes for Staphylococcus aureus enterotoxins, exfoliative toxins, toxic shock syndrome toxin 1, and methicillin resistance. J. Clin. Microbiol. 38:1032-1035.[Abstract/Free Full Text]
    31. 31
  31. Mellmann, A., A. W. Friedrich, N. Rosenkotter, J. Rothganger, H. Karch, R. Reintjes, and D. Harmsen. 2006. Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks. PLoS Med. 3:e33.[CrossRef][Medline]
    32. 32
  32. Moore, P. C., and J. A. Lindsay. 2001. Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes. J. Clin. Microbiol. 39:2760-2767.[Abstract/Free Full Text]
    33. 33
  33. Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.[Abstract/Free Full Text]
    34. 34
  34. Oliveira, D. C., C. Milheirico, S. Vinga, and H. de Lencastre. 2006. Assessment of allelic variation in the ccrAB locus in methicillin-resistant Staphylococcus aureus clones. J. Antimicrob. Chemother. 58:23-30.[Abstract/Free Full Text]
    35. 35
  35. Omoe, K., M. Ishikawa, Y. Shimoda, D. L. Hu, S. Ueda, and K. Shinagawa. 2002. Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates harboring seg, seh, or sei genes. J. Clin. Microbiol. 40:857-862.[Abstract/Free Full Text]
    36. 36
  36. Peacock, S. J., G. D. de Silva, A. Justice, A. Cowland, C. E. Moore, C. G. Winearls, and N. P. Day. 2002. Comparison of multilocus sequence typing and pulsed-field gel electrophoresis as tools for typing Staphylococcus aureus isolates in a microepidemiological setting. J. Clin. Microbiol. 40:3764-3770.[Abstract/Free Full Text]
    37. 37
  37. Ruppitsch, W., A. Indra, A. Stoger, B. Mayer, S. Stadlbauer, G. Wewalka, and F. Allerberger. 2006. Classifying spa types in complexes improves interpretation of typing results for methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 44:2442-2448.[Abstract/Free Full Text]
    38. 38
  38. Shopsin, B., M. Gomez, S. O. Montgomery, D. H. Smith, M. Waddington, D. E. Dodge, D. A. Bost, M. Riehman, S. Naidich, and B. N. Kreiswirth. 1999. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 37:3556-3563.[Abstract/Free Full Text]
    39. 39
  39. Shukla, S. K., M. E. Stemper, S. V. Ramaswamy, J. M. Conradt, R. Reich, E. A. Graviss, and K. D. Reed. 2004. Molecular characteristics of nosocomial and native American community-associated methicillin-resistant Staphylococcus aureus clones from rural Wisconsin. J. Clin. Microbiol. 42:3752-3757.[Abstract/Free Full Text]
    40. 40
  40. Stephens, A. J., F. Huygens, J. Inman-Bamber, E. P. Price, G. R. Nimmo, J. Schooneveldt, W. Munckhof, and P. M. Giffard. 2006. Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J. Med. Microbiol. 55:43-51.[CrossRef][Medline]
    41. 41
  41. Tang, Y. W., M. G. Waddington, D. H. Smith, J. M. Manahan, P. C. Kohner, L. M. Highsmith, H. Li, F. R. Cockerill III, R. L. Thompson, S. O. Montgomery, and D. H. Persing. 2000. Comparison of protein A gene sequencing with pulsed-field gel electrophoresis and epidemiologic data for molecular typing of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 38:1347-1351.[Abstract/Free Full Text]
    42. 42
  42. Tong, Z., F. Kong, B. Wang, X. Zeng, and G. L. Gilbert. 2007. A practical method for subtyping of Streptococcus agalactiae serotype III, of human origin, using rolling circle amplification. J. Microbiol. Methods 70:39-44.[CrossRef][Medline]
    43. 43
  43. Yamaguchi, T., K. Nishifuji, M. Sasaki, Y. Fudaba, M. Aepfelbacher, T. Takata, M. Ohara, H. Komatsuzawa, M. Amagai, and M. Sugai. 2002. Identification of the Staphylococcus aureus etd pathogenicity island which encodes a novel exfoliative toxin, ETD, and EDIN-B. Infect. Immun. 70:5835-5845.[Abstract/Free Full Text]
    44. 44
  44. Yamasaki, O., T. Yamaguchi, M. Sugai, C. Chapuis-Cellier, F. Arnaud, F. Vandenesch, J. Etienne, and G. Lina. 2005. Clinical manifestations of staphylococcal scalded-skin syndrome depend on serotypes of exfoliative toxins. J. Clin. Microbiol. 43:1890-1893.[Abstract/Free Full Text]

Journal of Clinical Microbiology, October 2007, p. 3302-3308, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01082-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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