Acta Univ. Agric. Silvic. Mendelianae Brun. 2013, 61(6), 1785-1792 | DOI: 10.11118/actaun201361061785

A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

Ivan Manga, Marcela Vyletělová
Agritec Plant Research, Ltd., Zemědělská 16, 787 01 Šumperk, Czech Republic

The Methicillin-resistant Staphylococcus aureus (MRSA) with the livestock-associated MRSA (LA-MRSA) are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confirmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour.

Keywords: RT-PCR, MRSA, S. aureus, livestock
Grants and funding:

This work was supported by projects of Ministry of Agriculture NAZV KUS QJ1210284.

Received: June 3, 2013; Published: November 24, 2013  Show citation

ACS AIP APA ASA Harvard Chicago IEEE ISO690 MLA NLM Turabian Vancouver
Manga, I., & Vyletělová, M. (2013). A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains. Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis, 61(6), 1785-1792. doi: 10.11118/actaun201361061785
Share...
Download citation
  • BibTeX (.bib)
  • Bookends (.ris)
  • EasyBib (.ris)
  • EndNote (.enw)
  • EndNote 8 (.xml)
  • ISI WoS (.isi)
  • Medlars (.medlars)
  • Mendeley (.ris)
  • MODS (.xml)
  • Papers (.ris)
  • RefWorks (.txt)
  • RefManager (.ris)
  • RIS (.ris)
  • MS Word (.xml)
  • Zotero (.ris)
    • Open full article

    References

    1. AHMADI, M., ROHANI, S. M. R., AYREMLOU, N., 2010: Detection of Staphylococcus aureus in milk by PCR. Comparative Clinical Pathology, 19, 1: 91-94. ISSN 1618-5641. DOI: 10.1007/s00580-009-0901-0 Go to original source...
    2. ALARCON, B., VICEDO, B., AZNAR, R., 2006: PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food. J Appl Microbiol., 100, 2: 352-364. ISSN 1365-2672. DOI: 10.1111/j.1365-2672.2005.02768.x Go to original source...
    3. ARBEFEVILLE, S. S., ZHANG, K., KROEGER, J. S., HOWARD, W. J., DIEKEMA, D. J., RICHTER, S. S., 2011: Prevalence and genetic relatedness of methicillin-susceptible Staphylococcus aureus isolates detected by the Xpert MRSA nasal assay. J Clin Microbiol., 49, 8: 2996-2999. ISSN 0095-1137. DOI: 10.1128/JCM.00046-11 Go to original source...
    4. AYDINER, A., LÜSEBRINK, J., SCHILDGEN, V., WINTERFELD, I., KNÜVER, O., SCHWARZ, K., MESSLER, S., SCHILDGEN, O., MATTNER, F., 2012: Comparison of two commercial PCR methods for methicillin-resistant Staphylococcus aureus (MRSA) screening in a tertiary care hospital. PLoS One. 7, 9: e43935. ISSN 1932-6203. DOI: 10.1371/journal.pone.0043935 Go to original source...
    5. CUNY, C., WITTE, W., 2005: PCR for the identification of methicillin-resistant Staphylococcus aureus (MRSA) strains using a single primer pair specific for SCCmec elements and the neighbouring chromosome-borne orfX. Clin Microbiol Infect., 11, 10: 834-837. ISSN 1469-0691. DOI: 10.1111/j.1469-0691.2005.01236.x Go to original source...
    6. DONNIO, P. Y. et al., 2007: Molecular and epidemiological evidence for spread of multiresistant methicillin-susceptible Staphylococcus aureus strains in hospitals. Antimicrob. Agents Chemother., 51, 12: 4342-4350. ISSN 1098-6596. DOI: 10.1128/AAC.01414-06 Go to original source...
    7. HEIN, I., LEHNER, A., RIECK, P., KLEIN, K., BRANDL, E., WAGNER, M., 2001: Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese. Appl Environ Microbiol., 67, 7: 3122-3126. ISSN 1098-5336. DOI: 10.1128/AEM.67.7.3122-3126.2001 Go to original source...
    8. 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 (SCCmec) typing of methicillin-resistant Staphylococcus aureus strains isolated in 11 Asian countries: a proposal for a new nomenclature for SCCmec elements. Antimicrob Agents Chemother., 50, 3: 1001-1012. ISSN 1098-6596. DOI: 10.1128/AAC.50.3.1001-1012.2006 Go to original source...
    9. IBRAHEM, S., 2010: Methicillin Resistance in Staphylococci: Horizontal Transfer of Mobile Genetic Element (SCCmec) between Staphylococcal Species, academic dissertation, National Institute for Health and Welfare. Helsinki: Helsinki University Print, 116 p. ISBN 978-952-245-309-9.
    10. KATAYAMA, Y., ROBINSON, D. A., ENRIGHT, M. C., CHAMBERS, H. F., 2005: Genetic background affects stability of mecA in Staphylococcus aureus. J Clin Microbiol., 43, 5: 2380-2383. ISSN 0095-1137. DOI: 10.1128/JCM.43.5.2380-2383.2005 Go to original source...
    11. KILIC, A., BASUSTAOGLU, A. C., 2011: Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles. Res. Microbiol., 162, 10: 1060-1066. ISSN 0923-2508. DOI: 10.1016/j.resmic.2011.07.009 Go to original source...
    12. KÖCK, R., SCHAUMBURG, F., MELLMANN, A., KÖKSAL, M., JURKE, A., BECKER, K., FRIEDRICH, A. W., 2013: Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) as causes of human infection and colonization in Germany. PLoS One. 8, 2: e55040. ISSN 1932-6203. DOI: 10.1371/journal.pone.0055040 Go to original source...
    13. LIVAK, K. J., SCHMITTGEN, T. D., 2001: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25, 4: 402-408. ISSN 1046-2023. DOI: 10.1006/meth.2001.1262 Go to original source...
    14. LUTEIJN, J. M., HUBBEN, G. A., PECHLIVANOGLOU, P., BONTEN, M. J., POSTMA, M. J., 2011: Diagnostic accuracy of culture-based and PCR-based detection tests for methicillin-resistant Staphylococcus aureus: a meta-analysis. Clin Microbiol Infect., 17, 2: 146-154. ISSN 1469-0691. DOI: 10.1111/j.1469-0691.2010.03202.x Go to original source...
    15. Clinical and Laboratory Standards Institute, 2007: Performance Standards for Antimicrobial Susceptibility Testing, M100-S17. CLSI, 940, Pennsylvania: Wayne, 172 p., ISBN 1-56238-625-5.
    16. POULSEN, A. B., SKOV, R., PALLESEN, L. V., 2003: Detection of methicillin resistance in coagulase-negative staphylococci and in staphylococci directly from simulated blood cultures using the EVIGENE MRSA Detection Kit. J. Antimicrob. Chemother., 51, 2: 419-421. ISSN 0305-7453. DOI: 10.1093/jac/dkg084 Go to original source...
    17. RUIMY, R., DOS-SANTOS, M., RASKINE, L., BERT, F., MASSON, R., ELBAZ, S., BONNAL, C., LUCET, J. C., LEFORT, A., FANTIN, B., WOLFF, M., HORNSTEIN, M., ANDREMONT, A., 2008: Accuracy and potential usefulness of triplex RT-PCR for improving antibiotic treatment of patients with blood cultures showing clustered gram-positive cocci on direct smears. J Clin Microbiol., 46, 6: 2045-2051. ISSN 0095-1137. DOI: 10.1128/JCM.02250-07 Go to original source...
    18. VANDERHAEGHEN, W., HERMANS, K., HAESEBROUCK, F., BUTAYE, P., 2010: Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals. Epidemiol. Infect., 138, 5: 606-625. ISSN 0950-2688. DOI: 10.1017/S0950268809991567 Go to original source...
    19. VAN DUIJKEREN, E., JANSEN, M. D., FLEMMING, S. C., DE NEELING, H. et al., 2007: Methicillin-resistant Staphylococcus aureus in pigs with exudative epidermitis. Emerg Infect Dis., 13, 9: 1408-1410. ISSN 1080-6059. DOI: 10.3201/eid1309.061268 Go to original source...
    20. WONG, H., LOUIE, L., LO, R. I. G, SIMOR, A. E., 2010: Characterization of Staphylococcus aureus Isolates with a Partial or Complete Absence of Staphylococcal Cassette Chromosome Elements. J. Clin. Microbiol., 48, 10: 3525. ISSN 0095-1137. DOI: 10.1128/JCM.00775-10 Go to original source...
    21. ZHANG, K., J. MCCLURE, J., ELSAYED, S., LOUIE, T., CONLY, J. M., 2008: Novel PCR assay for simultaneous identification of community-associated methicillin- resistant Staphylococcus aureus strains USA300 and USA400 and detection of mecA and Panton-Valentine leukocidin genes, with discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J. Clin. Microbiol., 46, 3: 1118-1122. ISSN 0095-1137. DOI: 10.1128/JCM.01309-07 Go to original source...

    This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY NC ND 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.


    Return to the content