Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples

Abstract

Staphylococcus epidermidis is considered to be one of the most common causes of nosocomial bloodstream infections, particularly in immune-compromised individuals. Here, we report the development and application of a novel peptide nucleic acid probe for the specific detection of S. epidermidis by fluorescence in situ hybridization. The theoretical estimates of probe matching specificity and sensitivity were 89 and 87%, respectively. More importantly, the probe was shown not to hybridize with closely related species such as Staphylococcus aureus. The method was subsequently successfully adapted for the detection of S. epidermidis in mixed-species blood cultures both by microscopy and flow cytometry.

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References

  1. Almeida C, Azevedo NF, Iversen C, Fanning S, Keevil CW, Vieira MJ (2009) Development and application of a novel peptide nucleic acid probe for the specific detection of Cronobacter (Enterobacter sakazakii) in powdered infant formula. Appl Environ Microbiol 75(9):2925–2930

    PubMed  Article  CAS  Google Scholar 

  2. Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ (2011) Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One 6(3):e14786

    PubMed  Article  CAS  Google Scholar 

  3. Ashelford KE, Weightman AJ, Fry JC (2002) PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Res 30:3481–3489

    PubMed  Article  CAS  Google Scholar 

  4. Barken KB, Haagensen JA, Tolker-Nielsen T (2007) Advances in nucleic acid-based diagnostics of bacterial infections. Clin Chim Acta 384:1–11

    PubMed  Article  CAS  Google Scholar 

  5. Cerqueira L, Azevedo NF, Almeida C, Jardim T, Keevil CW, Vieira MJ (2008) DNA mimics for the rapid identification of microorganisms by fluorescence in situ hybridization (FISH). Int J Mol Sci 9:1944–1960

    PubMed  Article  CAS  Google Scholar 

  6. Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, Garrity GM, Tiedje JM (2005) The ribosomal database project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33:D294–D296

    PubMed  Article  CAS  Google Scholar 

  7. Guimaraes N, Azevedo NF, Figueiredo C, Keevil CW, Vieira MJ (2007) Development and application of a novel peptide nucleic acid probe for the specific detection of Helicobacter pylori in gastric biopsy specimens. J Clin Microbiol 45:3089–3094

    PubMed  Article  CAS  Google Scholar 

  8. Haimi-Cohen Y, Vellozzi EM, Rubin LG (2002) Initial concentration of Staphylococcus epidermidis in simulated pediatric blood cultures correlates with time to positive results with the automated, continuously monitored BACTEC blood culture system. J Clin Microbiol 40:898–901

    PubMed  Article  Google Scholar 

  9. Hartmann H, Stender H, Schafer A, Autenrieth IB, Kempf VAJ (2005) Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry. J Clin Microbiol 43:4855–4857

    PubMed  Article  CAS  Google Scholar 

  10. Jukes L, Mikhail J, Bome-Mannathoko N, Hadfield SJ, Harris LG, El-Bouri K, Davies AP, Mack D (2010) Rapid differentiation of Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci and methicillin susceptibility testing directly from growth-positive blood cultures by multiplex real-time PCR. J Med Microbiol 59:1456–1461

    PubMed  Article  Google Scholar 

  11. Krimmer V, Merkert H, von Eiff C, Frosch M, Eulert J, Lohr JF, Hacker J, Ziebuhr W (1999) Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization. J Clin Microbiol 37:2667–2673

    PubMed  CAS  Google Scholar 

  12. Larsen MK, Thomsen TR, Moser C, Hoiby N, Nielsen PH (2008) Use of cultivation-dependent and -independent techniques to assess contamination of central venous catheters: a pilot study. BMC Clin Pathol 8:10

    PubMed  Article  Google Scholar 

  13. Markham NR, Zuker M (2005) DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res 33:W577–W581

    PubMed  Article  CAS  Google Scholar 

  14. McGinnis S, Madden TL (2004) BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res 32:W20–W25

    PubMed  Article  CAS  Google Scholar 

  15. Nielsen PE, Egholm M, Berg RH, Buchardt O (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254:1497–1500

    PubMed  Article  CAS  Google Scholar 

  16. Oliveira K, Haase G, Kurtzman C, Hyldig-Nielsen JJ, Stender H (2001) Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes. J Clin Microbiol 39:4138–4141

    PubMed  Article  CAS  Google Scholar 

  17. Perry-O’Keefe H, Rigby S, Oliveira K, Sorensen D, Stender H, Coull J, Hyldig-Nielsen JJ (2001) Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Meth 47:281–292

    Article  Google Scholar 

  18. Stender H, Lund K, Petersen KH, Rasmussen OF, Hongmanee P, Miorner H, Godtfredsen SE (1999) Fluorescence in situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of Mycobacterium cultures. J Clin Microbiol 37:2760–2765

    PubMed  CAS  Google Scholar 

  19. Stender H, Fiandaca M, Hyldig-Nielsen JJ, Coull J (2002) PNA for rapid microbiology. J Microbiol Meth 48:1–17

    Article  CAS  Google Scholar 

  20. Tavares A, Inacio J, Melo-Cristino J, Couto I (2008) Use of fluorescence in situ hybridization for rapid identification of staphylococci in blood culture samples collected in a portuguese hospital. J Clin Microbiol 46:3097–3100

    PubMed  Article  Google Scholar 

  21. Trebesius K, Panthel K, Strobel S, Vogt K, Faller G, Kirchner T, Kist M, Heesemann J, Haas R (2000) Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation. Gut 46:608–614

    PubMed  Article  CAS  Google Scholar 

  22. Vuong C, Otto M (2002) Staphylococcus epidermidis infections. Microbes Infect 4:481–489

    PubMed  Article  Google Scholar 

  23. Zakrzewskaczerwinska J, Gaszewskamastalarz A, Pulverer G, Mordarski M (1992) Identification of Staphylococcus epidermidis using a 16 s ribosomal-Rna-directed oligonucleotide probe. FEMS Microbiol Lett 100:51–58

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the Portuguese Institute Fundação para a Ciência e Tecnologia (PhD Fellowship SFRH/BD/29297/2006 and Post-Doc Fellowship SFRH/BPD/42208/2007).

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Correspondence to N. F. Azevedo.

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Azevedo, N.F., Jardim, T., Almeida, C. et al. Application of flow cytometry for the identification of Staphylococcus epidermidis by peptide nucleic acid fluorescence in situ hybridization (PNA FISH) in blood samples. Antonie van Leeuwenhoek 100, 463–470 (2011). https://doi.org/10.1007/s10482-011-9595-9

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Keywords

  • Staphylococcus epidermidis
  • Bloodstream infection
  • Flow cytometry
  • FISH
  • Nanodiagnostics
  • Permeabilization