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Volume 58, Issue 8

A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome (SCC) typing Free

Abstract

The aim of this study was to develop a new discriminatory method for MRSA SCC typing based on multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay to enable rapid identification and classification of MRSA SCC types in a clinical laboratory. Forty-five primer sets and 49 probes were designed and tested in uniplex PCR (uPCR) and mPCR/RLB. Probes were compared to 14 whole-genome sequences and 18 partial SCC gene sequences of and complete genome and partial SCC genes of seven non-MRSA strains, including meticillin-susceptible and meticillin-resistant coagulase-negative staphylococci. The method was tested on a set of 42 well-characterized reference MRSA strains. It identified all five epidemiologically relevant SCC types and 26 subtypes, including established and new subtypes of SCC III, IV (eight subtypes each) and V (three subtypes). The discriminatory power of mPCR/RLB SCC typing was similar to that of MLST and typing (Simpson indices of diversity of 0.916, 0.926 and 0.882, respectively; differences not statistically significant). The application of mPCR/RLB hybridization assay to MRSA SCC typing can improve the specificity, discriminatory power and throughput of the typing procedure. The detection of up to 43 mPCR products in a single hybridization assay transforms MRSA SCC typing from passive epidemiological library typing into a potential tool for near-real-time infection control surveillance and tracking of MRSA transmission in hospitals.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): MLST, multilocus sequence typing , mPCR/RLB, multiplex PCR-based reverse line-blot hybridization , MRSA, meticillin-resistant S. aureus , MSSA, meticillin-sensitive S. aureus , SCCmec, staphylococcal cassette chromosome mec and uPCR, uniplex PCR
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2009-08-01
2024-04-26
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References

  1. Aires de Sousa M., de Lencastre H. 2004; Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunol Med Microbiol 40:101–111 [CrossRef]
     [Google Scholar]
  2. Cai Y., Kong F., Wang Q., Tong Z., Sintchenko V., Zeng X., Gilbert G. L. 2007; Comparison of single- and multilocus sequence typing and toxin gene profiling for characterization of methicillin-resistant Staphylococcus aureus . J Clin Microbiol 45:3302–3308 [CrossRef]
     [Google Scholar]
  3. Chongtrakool P., Ito T., Ma X. X., Kondo Y., Trakulsomboon S., Tiensasitorn C., Jamklang M., Chavalit T., Song J. H., Hiramatsu K. 2006; Staphylococcal cassette chromosome mec (SCC mec ) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCC mec elements. Antimicrob Agents Chemother 50:1001–1012 [CrossRef]
     [Google Scholar]
  4. Coombs G. W., Nimmo G. R., Bell J. M., Huygens F., O'Brien F. G., Malkowski M. J., Pearson J. C., Stephens A. J., Giffard P. M. 2004; Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia. J Clin Microbiol 42:4735–4743 [CrossRef]
     [Google Scholar]
  5. Daum R. S., Ito T., Hiramatsu K., Hussain F., Mongkolrattanothai K., Jamklang M., Boyle-Vavra S. 2002; A novel methicillin-resistance cassette in community-acquired methicillin-resistant Staphylococcus aureus isolates of diverse genetic backgrounds. J Infect Dis 186:1344–1347 [CrossRef]
     [Google Scholar]
  6. Deurenberg R. H., Stobberingh E. E. 2008; The evolution of Staphylococcus aureus . Infect Genet Evol 8:747–763 [CrossRef]
     [Google Scholar]
  7. Grundmann H., Hori S., Tanner G. 2001; Determining confidence intervals when measuring genetic diversity and the discriminatory abilities of typing methods for microorganisms. J Clin Microbiol 39:4190–4192 [CrossRef]
     [Google Scholar]
  8. Hanssen A. M., Sollid J. U. 2007; Multiple staphylococcal cassette chromosomes and allelic variants of cassette chromosome recombinases in Staphylococcus aureus and coagulase-negative staphylococci from Norway. Antimicrob Agents Chemother 51:1671–1677 [CrossRef]
     [Google Scholar]
  9. Hunter P. R., Gaston M. A. 1988; Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26:2465–2466
     [Google Scholar]
  10. Huygens F., Stephens A. J., Nimmo G. R., Giffard P. M. 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 [CrossRef]
     [Google Scholar]
  11. Huygens F., Inman-Bamber J., Nimmo G. R., Munckhof W., Schooneveldt J., Harrison B., McMahon J. A., Giffard P. M. 2006; Staphylococcus aureus genotyping using novel real-time PCR formats. J Clin Microbiol 44:3712–3719 [CrossRef]
     [Google Scholar]
  12. Ito T., Hiramatsu K. 1998; Acquisition of methicillin resistance and progression of multiantibiotic resistance in methicillin-resistant Staphylococcus aureus . Yonsei Med J 39:526–533 [CrossRef]
     [Google Scholar]
  13. Ito T., Katayama Y., Hiramatsu K. 1999; Cloning and nucleotide sequence determination of the entire mec DNA of pre-methicillin-resistant Staphylococcus aureus N315. Antimicrob Agents Chemother 43:1449–1458
     [Google Scholar]
  14. Ito T., Katayama Y., Asada K., Mori N., Tsutsumimoto K., Tiensasitorn C., Hiramatsu K. 2001; Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus . Antimicrob Agents Chemother 45:1323–1336 [CrossRef]
     [Google Scholar]
  15. Ito T., Ma X. X., Takeuchi F., Okuma K., Yuzawa H., Hiramatsu K. 2004; Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC . Antimicrob Agents Chemother 48:2637–2651 [CrossRef]
     [Google Scholar]
  16. Kondo Y., Ito T., Ma X. X., Watanabe S., Kreiswirth B. N., Etienne J., Hiramatsu K. 2007; Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec , ccr , and major differences in junkyard regions. Antimicrob Agents Chemother 51:264–274 [CrossRef]
     [Google Scholar]
  17. Kong F., Gilbert G. L. 2006; Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) – a practical epidemiological and diagnostic tool. Nat Protoc 1:2668–2680
     [Google Scholar]
  18. Kong F., Ma L., Gilbert G. L. 2005; Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization. J Med Microbiol 54:1133–1138 [CrossRef]
     [Google Scholar]
  19. Kong F., Brown M., Sabananthan A., Zeng X., Gilbert G. L. 2006; Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes. J Clin Microbiol 44:1887–1891 [CrossRef]
     [Google Scholar]
  20. Lalani T., Federsoiel J. J., Boucher H. W., Rude T. H., Bae I.-G., Rybak M. J., Tonthat G. T., Corey G. R., Stryjewski M. E. other authors 2008; Associations between the genotypes of Staphylococcus aureus bloodstream isolates and clinical characteristics and outcomes of bacteremic patients. J Clin Microbiol 46:2890–2896 [CrossRef]
     [Google Scholar]
  21. Ma X. X., Ito T., Tiensasitorn C., Jamklang M., Chongtrakool P., Boyle-Vavra S., Daum R. S., Hiramatsu K. 2002; Novel type of staphylococcal cassette chromosome mec identified in community-acquired methicillin-resistant Staphylococcus aureus strains. Antimicrob Agents Chemother 46:1147–1152 [CrossRef]
     [Google Scholar]
  22. Milheirico C., Oliveira D. C., de Lencastre H. 2007; Update to the multiplex PCR strategy for assigning of mec element types in Staphylococcus aureus . Antimicrob Agents Chemother 51:3374–3377 [CrossRef]
     [Google Scholar]
  23. Mombach Pinheiro Machado A. B., Reiter K. C., Paiva R. M., Barth A. L. 2007; Distribution of staphylococcal cassette chromosome mec (SCC mec ) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microbiol 56:1328–1333 [CrossRef]
     [Google Scholar]
  24. Oliveira D. C., de Lencastre H. 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 [CrossRef]
     [Google Scholar]
  25. Oliveira D. C., Milheirico C., de Lencastre H. 2006a; Redefining a structural variant of staphylococcal cassette chromosome mec, SCC mec type VI. Antimicrob Agents Chemother 50:3457–3459 [CrossRef]
     [Google Scholar]
  26. Oliveira D. C., Milheirico C., Vinga S., de Lencastre H. 2006b; Assessment of allelic variation in the ccrAB locus in methicillin-resistant Staphylococcus aureus clones. J Antimicrob Chemother 58:23–30 [CrossRef]
     [Google Scholar]
  27. Shore A. C., Rossney A. S., O'Connell B., Herra C. M., Sullivan D. J., Humphreys H., Coleman D. C. 2008; Detection of staphylococcal cassette chromosome mec-associated DNA segments in multiresistant methicillin-susceptible Staphylococcus aureus (MSSA) and identification of Staphylococcus epidermidis ccrAB4 in both methicillin-resistant S. aureus and MSSA. Antimicrob Agents Chemother 52:4407–4419 [CrossRef]
     [Google Scholar]
  28. Stephens A. J., Huygens F., Inman-Bamber J., Price E. P., Nimmo G. R., Schooneveldt J., Munckhof W., Giffard P. M. 2006; Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J Med Microbiol 55:43–51 [CrossRef]
     [Google Scholar]
  29. Stephens A. J., Huygens F., Giffard P. M. 2007; Systematic derivation of marker sets for staphylococcal cassette chromosome mec typing. Antimicrob Agents Chemother 51:2954–2964 [CrossRef]
     [Google Scholar]
  30. Takano T., Higuchi W., Otsuka T., Baranovich T., Enany S., Saito K., Isobe H., Dohmae S., Ozaki K. other authors 2008; Novel characteristics of community-acquired methicillin-resistant Staphylococcus aureus strains belonging to multilocus sequence type 59 in Taiwan. Antimicrob Agents Chemother 52:837–845 [CrossRef]
     [Google Scholar]
  31. Wang Q., Kong F., Jelfs P., Gilbert G. L. 2008; Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization. J Med Microbiol 57:827–838 [CrossRef]
     [Google Scholar]
  32. Zhang K., McClure J. A., Elsayed S., Louie T., Conly J. M. 2005; Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus . J Clin Microbiol 43:5026–5033 [CrossRef]
     [Google Scholar]
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A new multiplex PCR-based reverse line-blot hybridization (mPCR/RLB) assay for rapid staphylococcal cassette chromosome mec (SCCmec) typing
J. Med. Microbiol. 58, 1045 (2009); https://doi.org/10.1099/jmm.0.007955-0
/content/journal/jmm/10.1099/jmm.0.007955-0
/content/journal/jmm/10.1099/jmm.0.007955-0
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