1887

Abstract

The genetic diversity between major meticillin-resistant (MRSA) lineages was probed using fluorescent amplified fragment length polymorphism (FAFLP) as a random genome sampling tool. Genomic DNA was digested with endonucleases II and 6I and a subset of the restricted fragments were amplified using the primer pair II+A and 6I+0. Sixty-seven FAFLP profiles consisting of 46–68 amplified fragments ranging in size from 50 to 600 bp were exhibited amongst the 71 isolates analysed. Cluster analysis of FAFLP data revealed concordance with typing and MLST clonal complexes (CC), with isolates of each CC grouping in the same FAFLP cluster. Furthermore, FAFLP could differentiate subtypes within the homogeneous CC22 isolates and also between MLST sequence types 8 and 239. The discriminatory power of FAFLP was 0.998 compared to values of 0.975 and 0.909 for typing and MLST, respectively. Thus, FAFLP analysis proved to be a rapid, reproducible and high-resolution tool that displayed the microheterogeneity within MRSA lineages. Using FAFLP data, lineage-specific fragments were identified and sequenced; these encoded toxins, antibiotic resistance determinants and bacteriophage resistance factors. Lineage-specific sequence variations were observed, which may provide insights into the evolution and fitness of successful lineages. This will also aid in the development of rapid and high-throughput diagnostic PCR-based assays for the identification of MRSA lineages in resource-poor settings.

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2012-12-01
2020-01-26
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References

  1. Chen L. , Mediavilla J. R. , Oliveira D. C. , Willey B. M. , de Lencastre H. , Kreiswirth B. N. . ( 2009; ). Multiplex real-time PCR for rapid staphylococcal cassette chromosome mec typing. . J Clin Microbiol 47:, 3692–3706. [CrossRef] [PubMed]
    [Google Scholar]
  2. Cosgrove S. E. , Sakoulas G. , Perencevich E. N. , Schwaber M. J. , Karchmer A. W. , Carmeli Y. . ( 2003; ). Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. . Clin Infect Dis 36:, 53–59. [CrossRef] [PubMed]
    [Google Scholar]
  3. Dailey F. E. , Cronan J. E. Jr . ( 1986; ). Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source. . J Bacteriol 165:, 453–460.[PubMed]
    [Google Scholar]
  4. Desai M. , Tanna A. , Wall R. , Efstratiou A. , George R. , Stanley J. . ( 1998; ). Fluorescent amplified-fragment length polymorphism analysis of an outbreak of group A streptococcal invasive disease. . J Clin Microbiol 36:, 3133–3137.[PubMed]
    [Google Scholar]
  5. Engelberg-Kulka H. , Glaser G. . ( 1999; ). Addiction modules and programmed cell death and antideath in bacterial cultures. . Annu Rev Microbiol 53:, 43–70. [CrossRef] [PubMed]
    [Google Scholar]
  6. Enright M. C. . ( 2003; ). The evolution of a resistant pathogen – the case of MRSA. . Curr Opin Pharmacol 3:, 474–479. [CrossRef] [PubMed]
    [Google Scholar]
  7. Enright M. C. , Day N. P. J. , Davies C. E. , Peacock S. J. , Spratt B. G. . ( 2000; ). Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . . J Clin Microbiol 38:, 1008–1015.[PubMed]
    [Google Scholar]
  8. Enright M. C. , Robinson D. A. , Randle G. , Feil E. J. , Grundmann H. , Spratt B. G. . ( 2002; ). The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). . Proc Natl Acad Sci U S A 99:, 7687–7692. [CrossRef] [PubMed]
    [Google Scholar]
  9. Fitzgerald J. R. , Sturdevant D. E. , Mackie S. M. , Gill S. R. , Musser J. M. . ( 2001; ). Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. . Proc Natl Acad Sci U S A 98:, 8821–8826. [CrossRef] [PubMed]
    [Google Scholar]
  10. Frankel M. B. , Wojcik B. M. , DeDent A. C. , Missiakas D. M. , Schneewind O. . ( 2010; ). ABI domain-containing proteins contribute to surface protein display and cell division in Staphylococcus aureus . . Mol Microbiol 78:, 238–252.[PubMed]
    [Google Scholar]
  11. Garvey P. , van Sinderen D. , Twomey D. , Hill C. , Fitzgerald G. F. . ( 1995; ). Molecular genetics of bacteriophage and natural phage defence systems in the genus Lactococcus . . Int Dairy J 5:, 905–947. [CrossRef]
    [Google Scholar]
  12. Grady R. , Desai M. , O’Neill G. , Cookson B. , Stanley J. . ( 1999; ). Genotyping of epidemic methicillin-resistant Staphylococcus aureus phage type 15 isolates by fluorescent amplified-fragment length polymorphism analysis. . J Clin Microbiol 37:, 3198–3203.[PubMed]
    [Google Scholar]
  13. Grady R. , O’Neill G. , Cookson B. , Stanley J. . ( 2000; ). Fluorescent amplified-fragment length polymorphism analysis of the MRSA epidemic. . FEMS Microbiol Lett 187:, 27–30. [CrossRef] [PubMed]
    [Google Scholar]
  14. 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] [PubMed]
    [Google Scholar]
  15. Grundmann H. , Aanensen D. M. , van den Wijngaard C. C. , Spratt B. G. , Harmsen D. , Friedrich A. W. . for the European Staphylococcal Reference Laboratory Working Group ( 2010; ). Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. . PLoS Med 7:, e1000215. [CrossRef] [PubMed]
    [Google Scholar]
  16. 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.[PubMed]
    [Google Scholar]
  17. Inoue J. , Honda M. , Ikawa S. , Shibata T. , Mikawa T. . ( 2008; ). The process of displacing the single-stranded DNA-binding protein from single-stranded DNA by RecO and RecR proteins. . Nucleic Acids Res 36:, 94–109. [CrossRef] [PubMed]
    [Google Scholar]
  18. Janssen P. , Coopman R. , Huys G. , Swings J. , Bleeker M. , Vos P. , Zabeau M. , Kersters K. . ( 1996; ). Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. . Microbiology 142:, 1881–1893. [CrossRef] [PubMed]
    [Google Scholar]
  19. Kober M. V. , Abreu M. B. , Bogo M. R. , Ferreira C. A. S. , Oliveira S. D. . ( 2011; ). Differentiation of Salmonella enteritidis isolates by fluorescent amplified fragment length polymorphism. . Foodborne Pathog Dis 8:, 19–26. [CrossRef] [PubMed]
    [Google Scholar]
  20. Lindsay J. A. , Holden M. T. G. . ( 2004; ). Staphylococcus aureus: superbug, super genome?. Trends Microbiol 12:, 378–385. [CrossRef] [PubMed]
    [Google Scholar]
  21. Lindsay J. A. , Moore C. E. , Day N. P. , Peacock S. J. , Witney A. A. , Stabler R. A. , Husain S. E. , Butcher P. D. , Hinds J. . ( 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. [CrossRef] [PubMed]
    [Google Scholar]
  22. Malachowa N. , DeLeo F. R. . ( 2010; ). Mobile genetic elements of Staphylococcus aureus . . Cell Mol Life Sci 67:, 3057–3071. [CrossRef] [PubMed]
    [Google Scholar]
  23. Melles D. C. , Gorkink R. F. J. , Boelens H. A. M. , Snijders S. V. , Peeters J. K. , Moorhouse M. J. , van der Spek P. J. , van Leeuwen W. B. , Simons G. . & other authors ( 2004; ). Natural population dynamics and expansion of pathogenic clones of Staphylococcus aureus . . J Clin Invest 114:, 1732–1740.[PubMed] [CrossRef]
    [Google Scholar]
  24. Melles D. C. , Schouls L. , François P. , Herzig S. , Verbrugh H. A. , van Belkum A. , Schrenzel J. . ( 2009; ). High-throughput typing of Staphylococcus aureus by amplified fragment length polymorphism (AFLP) or multi-locus variable number of tandem repeat analysis (MLVA) reveals consistent strain relatedness. . Eur J Clin Microbiol Infect Dis 28:, 39–45. [CrossRef] [PubMed]
    [Google Scholar]
  25. Milheiriço C. , Oliveira D. C. , de Lencastre H. . ( 2007; ). Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus . . Antimicrob Agents Chemother 51:, 3374–3377. [CrossRef] [PubMed]
    [Google Scholar]
  26. Musser J. M. , Kapur V. . ( 1992; ). Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. . J Clin Microbiol 30:, 2058–2063.[PubMed]
    [Google Scholar]
  27. Noto M. J. , Kreiswirth B. N. , Monk A. B. , Archer G. L. . ( 2008; ). Gene acquisition at the insertion site for SCCmec, the genomic island conferring methicillin resistance in Staphylococcus aureus . . J Bacteriol 190:, 1276–1283. [CrossRef] [PubMed]
    [Google Scholar]
  28. Platt S. , Pichon B. , George R. , Green J. . ( 2006; ). A bioinformatics pipeline for high-throughput microbial multilocus sequence typing (MLST) analyses. . Clin Microbiol Infect 12:, 1144–1146. [CrossRef] [PubMed]
    [Google Scholar]
  29. Robinson D. A. , Enright M. C. . ( 2003; ). Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus . . Antimicrob Agents Chemother 47:, 3926–3934. [CrossRef] [PubMed]
    [Google Scholar]
  30. Robinson D. A. , Enright M. C. . ( 2004; ). Evolution of Staphylococcus aureus by large chromosomal replacements. . J Bacteriol 186:, 1060–1064. [CrossRef] [PubMed]
    [Google Scholar]
  31. Rozen S. , Skaletsky H. . ( 2000; ). Primer3 on the WWW for general users and for biologist programmers. . In Bioinformatics Methods and Protocols: Methods in Molecular Biology, pp. 365–386. Edited by Krawetz S. , Misener S. . , Totowa, NJ:: Humana Press;. [CrossRef]
    [Google Scholar]
  32. Sancar A. . ( 2003; ). Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. . Chem Rev 103:, 2203–2237. [CrossRef] [PubMed]
    [Google Scholar]
  33. Savelkoul P. H. M. , Melles D. C. , Buffing N. , Gorkink R. , Simons G. , van Belkum A. . ( 2007; ). High density whole genome fingerprinting of methicillin-resistant and -susceptible strains of Staphylococcus aureus in search of phenotype-specific molecular determinants. . J Microbiol Methods 71:, 44–54. [CrossRef] [PubMed]
    [Google Scholar]
  34. Shopsin B. , Gomez M. , Montgomery S. O. , Smith D. H. , Waddington M. , Dodge D. E. , Bost D. A. , Riehman M. , Naidich S. , Kreiswirth B. N. . ( 1999; ). Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. . J Clin Microbiol 37:, 3556–3563.[PubMed]
    [Google Scholar]
  35. Simpson E. H. . ( 1949; ). Measurement of diversity. . Nature 163:, 688. [CrossRef]
    [Google Scholar]
  36. Underwood A. . ( 2011; ). ALFIE – AFLP Fragment Predictor Program. HPA online bioinformatic tools. Retrieved July 22, 2011, from http://www.hpa-bioinformatics.org.uk/cgi-bin/ALFIE/index.cgi.
  37. van Leeuwen W. B. , Melles D. C. , Alaidan A. , Al-Ahdal M. , Boelens H. A. M. , Snijders S. V. , Wertheim H. , van Duijkeren E. , Peeters J. K. . & other authors ( 2005; ). Host- and tissue-specific pathogenic traits of Staphylococcus aureus . . J Bacteriol 187:, 4584–4591. [CrossRef] [PubMed]
    [Google Scholar]
  38. Vos P. , Hogers R. , Bleeker M. , Reijans M. , van de Lee T. , Hornes M. , Frijters A. , Pot J. , Peleman J. . & other authors ( 1995; ). AFLP: a new technique for DNA fingerprinting. . Nucleic Acids Res 23:, 4407–4414. [CrossRef] [PubMed]
    [Google Scholar]
  39. Yoon H. J. , Choi J. Y. , Kim C. O. , Kim J. M. , Song Y. G. . ( 2005; ). A comparison of clinical features and mortality among methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus endocarditis. . Yonsei Med J 46:, 496–502. [CrossRef] [PubMed]
    [Google Scholar]
  40. Zetola N. , Francis J. S. , Nuermberger E. L. , Bishai W. R. . ( 2005; ). Community-acquired meticillin-resistant Staphylococcus aureus: an emerging threat. . Lancet Infect Dis 5:, 275–286. [CrossRef] [PubMed]
    [Google Scholar]
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