1887

Abstract

Purpose. Staphylococcus epidermidis colonies often display several morphologies and antimicrobial susceptibility patterns when cultured from device-related infections, and may represent one or multiple genotypes. Genotyping may be helpful in the clinical interpretation, but is time consuming and expensive. We wanted to establish a method for rapid discrimination of S. epidermidis genotypes for use in a routine microbiology laboratory.

Methodology. A real-time PCR targeting eight discriminatory class I or II single-nucleotide polymorphisms (SNPs) in six of the seven housekeeping genes was constructed. Post PCR, high-resolution melt (HRM) analysis using EvaGreen as fluorophore discriminated amplicons based on their percentage GC content.

Results. In silico, 42 representative sequence types (STs), including all major MLST group and subgroup founders, were separated into 23 different cluster profiles with a Simpson’s index of diversity of 0.97. By HRM-PCR, 11 commonly encountered hospital and outbreak STs were separated into eight HRM patterns.

Conclusion. This method can rapidly establish whether S. epidermidis strains belong to different genotypes. It can be used in patients with S. epidermidis infections, as an aid in outbreak investigations and to select strains for investigation with more discriminatory methods, saving workload and costs. Results may be obtained the same day as culture results. Its strength lies mainly in indicating differences, as some STs may have the same melt profile. Changes in S. epidermidis epidemiology may warrant alterations in the inclusion of SNPs. We believe this method can reduce the threshold for performing genotyping analysis on an increasingly important nosocomial pathogen.

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2018-01-02
2019-12-08
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References

  1. Widerström M, Wiström J, Sjöstedt A, Monsen T. Coagulase- negative staphylococci: update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus. Eur J Clin Microbiol Infect Dis 2012;31:7–20 [CrossRef][PubMed]
    [Google Scholar]
  2. Otto M. Staphylococcus epidermidis–the 'accidental' pathogen. Nat Rev Microbiol 2009;7:555–567 [CrossRef][PubMed]
    [Google Scholar]
  3. Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27:870–926 [CrossRef][PubMed]
    [Google Scholar]
  4. Miragaia M, Couto I, Pereira SF, Kristinsson KG, Westh H et al. Molecular characterization of methicillin-resistant Staphylococcus epidermidis clones: evidence of geographic dissemination. J Clin Microbiol 2002;40:430–438 [CrossRef][PubMed]
    [Google Scholar]
  5. Kozitskaya S, Olson ME, Fey PD, Witte W, Ohlsen K et al. Clonal analysis of Staphylococcus epidermidis isolates carrying or lacking biofilm-mediating genes by multilocus sequence typing. J Clin Microbiol 2005;43:4751–4757 [CrossRef][PubMed]
    [Google Scholar]
  6. Schoenfelder SM, Lange C, Eckart M, Hennig S, Kozytska S et al. Success through diversity - how Staphylococcus epidermidis establishes as a nosocomial pathogen. Int J Med Microbiol 2010;300:380–386 [CrossRef][PubMed]
    [Google Scholar]
  7. Widerström M, Monsen T, Karlsson C, Edebro H, Johansson A et al. Clonality among multidrug-resistant hospital-associated Staphylococcus epidermidis in northern Europe. Scand J Infect Dis 2009;41:642–649 [CrossRef][PubMed]
    [Google Scholar]
  8. Gordon RJ, Miragaia M, Weinberg AD, Lee CJ, Rolo J et al. Staphylococcus epidermidis colonization is highly clonal across US cardiac centers. J Infect Dis 2012;205:1391–1398 [CrossRef][PubMed]
    [Google Scholar]
  9. Rolo J, de Lencastre H, Miragaia M. Strategies of adaptation of Staphylococcus epidermidis to hospital and community: amplification and diversification of SCCmec. J Antimicrob Chemother 2012;67:1333–1341 [CrossRef][PubMed]
    [Google Scholar]
  10. Cherifi S, Byl B, Deplano A, Nagant C, Nonhoff C et al. Genetic characteristics and antimicrobial resistance of Staphylococcus epidermidis isolates from patients with catheter-related bloodstream infections and from colonized healthcare workers in a Belgian hospital. Ann Clin Microbiol Antimicrob 2014;13:20 [CrossRef][PubMed]
    [Google Scholar]
  11. Galdbart JO, Morvan A, Desplaces N, El Solh N. Phenotypic and genomic variation among Staphylococcus epidermidis strains infecting joint prostheses. J Clin Microbiol 1999;37:1306–1312[PubMed]
    [Google Scholar]
  12. Tande AJ, Osmon DR, Greenwood-Quaintance KE, Mabry TM, Hanssen AD et al. Clinical characteristics and outcomes of prosthetic joint infection caused by small colony variant staphylococci. MBio 2014;5:e01910-14 [CrossRef][PubMed]
    [Google Scholar]
  13. Nilsdotter-Augustinsson A, Koskela A, Ohman L, Söderquist B. Characterization of coagulase-negative staphylococci isolated from patients with infected hip prostheses: use of phenotypic and genotypic analyses, including tests for the presence of the ica operon. Eur J Clin Microbiol Infect Dis 2007;26:255–265 [CrossRef][PubMed]
    [Google Scholar]
  14. Maduka-Ezeh AN, Greenwood-Quaintance KE, Karau MJ, Berbari EF, Osmon DR et al. Antimicrobial susceptibility and biofilm formation of Staphylococcus epidermidis small colony variants associated with prosthetic joint infection. Diagn Microbiol Infect Dis 2012;74:224–229 [CrossRef][PubMed]
    [Google Scholar]
  15. Miragaia M, Carriço JA, Thomas JC, Couto I, Enright MC et al. Comparison of molecular typing methods for characterization of Staphylococcus epidermidis: proposal for clone definition. J Clin Microbiol 2008;46:118–129 [CrossRef][PubMed]
    [Google Scholar]
  16. Miragaia M, Thomas JC, Couto I, Enright MC, de Lencastre H. Inferring a population structure for Staphylococcus epidermidis from multilocus sequence typing data. J Bacteriol 2007;189:2540–2552 [CrossRef][PubMed]
    [Google Scholar]
  17. Jamaluddin TZ, Kuwahara-Arai K, Hisata K, Terasawa M, Cui L et al. Extreme genetic diversity of methicillin-resistant Staphylococcus epidermidis strains disseminated among healthy Japanese children. J Clin Microbiol 2008;46:3778–3783 [CrossRef][PubMed]
    [Google Scholar]
  18. Hellmark B, Söderquist B, Unemo M, Nilsdotter-Augustinsson Å. Comparison of Staphylococcus epidermidis isolated from prosthetic joint infections and commensal isolates in regard to antibiotic susceptibility, agr type, biofilm production, and epidemiology. Int J Med Microbiol 2013;303:32–39 [CrossRef][PubMed]
    [Google Scholar]
  19. Ahlstrand E, Hellmark B, Svensson K, Söderquist B. Long-term molecular epidemiology of Staphylococcus epidermidis blood culture isolates from patients with hematological malignancies. PLoS One 2014;9:e99045 [CrossRef][PubMed]
    [Google Scholar]
  20. Mendes RE, Deshpande LM, Costello AJ, Farrell DJ. Molecular epidemiology of Staphylococcus epidermidis clinical isolates from U.S. hospitals. Antimicrob Agents Chemother 2012;56:4656–4661 [CrossRef][PubMed]
    [Google Scholar]
  21. Conlan S, Mijares LA, Becker J, Blakesley RW, Bouffard GG et al. Staphylococcus epidermidis pan-genome sequence analysis reveals diversity of skin commensal and hospital infection-associated isolates. Genome Biol 2012;13:R64 [CrossRef][PubMed]
    [Google Scholar]
  22. Méric G, Miragaia M, de Been M, Yahara K, Pascoe B et al. Ecological overlap and horizontal gene transfer in Staphylococcus aureus and Staphylococcus epidermidis. Genome Biol Evol 2015;7:1313–1328 [CrossRef][PubMed]
    [Google Scholar]
  23. Harris LG, Murray S, Pascoe B, Bray J, Meric G et al. Biofilm morphotypes and population structure among Staphylococcus epidermidis from commensal and clinical samples. PLoS One 2016;11:e0151240 [CrossRef][PubMed]
    [Google Scholar]
  24. Sabat AJ, Budimir A, Nashev D, Sá-Leão R, van Dijl J et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Euro Surveill 2013;18:20380 [CrossRef][PubMed]
    [Google Scholar]
  25. Tolo I, Thomas JC, Fischer RS, Brown EL, Gray BM et al. Do Staphylococcus epidermidis genetic clusters predict isolation sources?. J Clin Microbiol 2016;54:1711–1719 [CrossRef][PubMed]
    [Google Scholar]
  26. Thomas JC, Zhang L, Robinson DA. Differing lifestyles of Staphylococcus epidermidis as revealed through Bayesian clustering of multilocus sequence types. Infect Genet Evol 2014;22:257–264 [CrossRef][PubMed]
    [Google Scholar]
  27. Robertson GA, Thiruvenkataswamy V, Shilling H, Price EP, Huygens F et al. Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases. J Med Microbiol 2004;53:35–45 [CrossRef][PubMed]
    [Google Scholar]
  28. Price EP, Inman-Bamber J, Thiruvenkataswamy V, Huygens F, Giffard PM. Computer-aided identification of polymorphism sets diagnostic for groups of bacterial and viral genetic variants. BMC Bioinformatics 2007;8:278 [CrossRef][PubMed]
    [Google Scholar]
  29. Stephens AJ, Huygens F, Inman-Bamber J, Price EP, Nimmo GR et al. Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J Med Microbiol 2006;55:43–51 [CrossRef][PubMed]
    [Google Scholar]
  30. Whiley DM, Goire N, Rahimi F, Lahra MM, Limnios AE et al. Real-time PCR genotyping of Neisseria gonorrhoeae isolates using 14 informative single nucleotide polymorphisms on gonococcal housekeeping genes. J Antimicrob Chemother 2013;68:322–328 [CrossRef][PubMed]
    [Google Scholar]
  31. Price EP, Thiruvenkataswamy V, Mickan L, Unicomb L, Rios RE et al. Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing. J Med Microbiol 2006;55:1061–1070 [CrossRef][PubMed]
    [Google Scholar]
  32. Lilliebridge RA, Tong SY, Giffard PM, Holt DC. The utility of high-resolution melting analysis of SNP nucleated PCR amplicons–an MLST based Staphylococcus aureus typing scheme. PLoS One 2011;6:e19749 [CrossRef][PubMed]
    [Google Scholar]
  33. Tong SY, Xie S, Richardson LJ, Ballard SA, Dakh F et al. High- resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms. PLoS One 2011;6:e29189 [CrossRef][PubMed]
    [Google Scholar]
  34. Andersson P, Tong SY, Bell JM, Turnidge JD, Giffard PM. Minim typing–a rapid and low cost MLST based typing tool for Klebsiella pneumoniae. PLoS One 2012;7:e33530 [CrossRef][PubMed]
    [Google Scholar]
  35. Widerström M. Significance of Staphylococcus epidermidis in health care-associated infections, from contaminant to clinically relevant pathogen: this is a wake-up call!. J Clin Microbiol 2016;54:1679–1681 [CrossRef][PubMed]
    [Google Scholar]
  36. Kahl BC, Becker K, Löffler B. Clinical significance and pathogenesis of Staphylococcal small colony variants in persistent infections. Clin Microbiol Rev 2016;29:401–427 [CrossRef][PubMed]
    [Google Scholar]
  37. van Eldere J, Peetermans WE, Struelens M, Deplano A, Bobbaers H. Polyclonal staphylococcal endocarditis caused by genetic variability. Clin Infect Dis 2000;31:24–30 [CrossRef][PubMed]
    [Google Scholar]
  38. Berg ES, Nolan T. High-resolution melt analysis. In Nolan T, Bustin SA. (editors) PCR Technology: Current Innovations Boca Raton, FL: CRC press, Taylor & Francis Group; 2013
    [Google Scholar]
  39. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 1988;26:2465–2466[PubMed]
    [Google Scholar]
  40. Tolo I, Thomas JC, Fischer RS, Brown EL, Gray BM et al. Do Staphylococcus epidermidis genetic clusters predict isolation sources?. J Clin Microbiol 2016;54:1711–1719 [CrossRef][PubMed]
    [Google Scholar]
  41. Lefever S, Pattyn F, Hellemans J, Vandesompele J. Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays. Clin Chem 2013;59:1470–1480 [CrossRef][PubMed]
    [Google Scholar]
  42. Taylor S, Scott R, Kurtz R, Fisher C, Patel V et al. A Practical Guide to High Resolution Melt Analysis Genotyping Bulletin 6004 Hercules, CA: Bio-Rad Laboratories, Inc; 2010
    [Google Scholar]
  43. Tong SY, Giffard PM. Microbiological applications of high-resolution melting analysis. J Clin Microbiol 2012;50:3418–3421 [CrossRef][PubMed]
    [Google Scholar]
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