Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 Jul;32(7):1768–1772. doi: 10.1128/jcm.32.7.1768-1772.1994

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory.

D J Geha 1, J R Uhl 1, C A Gustaferro 1, D H Persing 1
PMCID: PMC263789  PMID: 7929772

Abstract

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

Full text

PDF
1771

Images in this article

1770Image
on p.1770

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Archer G. L. Antimicrobial susceptibility and selection of resistance among Staphylococcus epidermidis isolates recovered from patients with infections of indwelling foreign devices. Antimicrob Agents Chemother. 1978 Sep;14(3):353–359. doi: 10.1128/aac.14.3.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Archer G. L., Pennell E. Detection of methicillin resistance in staphylococci by using a DNA probe. Antimicrob Agents Chemother. 1990 Sep;34(9):1720–1724. doi: 10.1128/aac.34.9.1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bej A. K., McCarty S. C., Atlas R. M. Detection of coliform bacteria and Escherichia coli by multiplex polymerase chain reaction: comparison with defined substrate and plating methods for water quality monitoring. Appl Environ Microbiol. 1991 Aug;57(8):2429–2432. doi: 10.1128/aem.57.8.2429-2432.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger-Bächi B., Barberis-Maino L., Strässle A., Kayser F. H. FemA, a host-mediated factor essential for methicillin resistance in Staphylococcus aureus: molecular cloning and characterization. Mol Gen Genet. 1989 Oct;219(1-2):263–269. doi: 10.1007/BF00261186. [DOI] [PubMed] [Google Scholar]
  5. Chambers H. F., Archer G., Matsuhashi M. Low-level methicillin resistance in strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1989 Apr;33(4):424–428. doi: 10.1128/aac.33.4.424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chambers H. F., Hartman B. J., Tomasz A. Increased amounts of a novel penicillin-binding protein in a strain of methicillin-resistant Staphylococcus aureus exposed to nafcillin. J Clin Invest. 1985 Jul;76(1):325–331. doi: 10.1172/JCI111965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chambers H. F., Sachdeva M. Binding of beta-lactam antibiotics to penicillin-binding proteins in methicillin-resistant Staphylococcus aureus. J Infect Dis. 1990 Jun;161(6):1170–1176. doi: 10.1093/infdis/161.6.1170. [DOI] [PubMed] [Google Scholar]
  8. Christensen G. D., Bisno A. L., Parisi J. T., McLaughlin B., Hester M. G., Luther R. W. Nosocomial septicemia due to multiply antibiotic-resistant Staphylococcus epidermidis. Ann Intern Med. 1982 Jan;96(1):1–10. doi: 10.7326/0003-4819-96-1-1. [DOI] [PubMed] [Google Scholar]
  9. Espy M. J., Smith T. F., Persing D. H. Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition. J Clin Microbiol. 1993 Sep;31(9):2361–2365. doi: 10.1128/jcm.31.9.2361-2365.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gerberding J. L., Miick C., Liu H. H., Chambers H. F. Comparison of conventional susceptibility tests with direct detection of penicillin-binding protein 2a in borderline oxacillin-resistant strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1991 Dec;35(12):2574–2579. doi: 10.1128/aac.35.12.2574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hartman B. J., Tomasz A. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1986 Jan;29(1):85–92. doi: 10.1128/aac.29.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hartman B. J., Tomasz A. Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus. J Bacteriol. 1984 May;158(2):513–516. doi: 10.1128/jb.158.2.513-516.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiramatsu K., Suzuki E., Takayama H., Katayama Y., Yokota T. Role of penicillinase plasmids in the stability of the mecA gene in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1990 Apr;34(4):600–604. doi: 10.1128/aac.34.4.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ilstrup D. M. Statistical methods in microbiology. Clin Microbiol Rev. 1990 Jul;3(3):219–226. doi: 10.1128/cmr.3.3.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Isaacs S. T., Tessman J. W., Metchette K. C., Hearst J. E., Cimino G. D. Post-PCR sterilization: development and application to an HIV-1 diagnostic assay. Nucleic Acids Res. 1991 Jan 11;19(1):109–116. doi: 10.1093/nar/19.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Karchmer A. W., Archer G. L., Dismukes W. E. Staphylococcus epidermidis causing prosthetic valve endocarditis: microbiologic and clinical observations as guides to therapy. Ann Intern Med. 1983 Apr;98(4):447–455. doi: 10.7326/0003-4819-98-4-447. [DOI] [PubMed] [Google Scholar]
  17. Ligozzi M., Rossolini G. M., Tonin E. A., Fontana R. Nonradioactive DNA probe for detection of gene for methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1991 Mar;35(3):575–578. doi: 10.1128/aac.35.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Madiraju M. V., Brunner D. P., Wilkinson B. J. Effects of temperature, NaCl, and methicillin on penicillin-binding proteins, growth, peptidoglycan synthesis, and autolysis in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Nov;31(11):1727–1733. doi: 10.1128/aac.31.11.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maidhof H., Reinicke B., Blümel P., Berger-Bächi B., Labischinski H. femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. J Bacteriol. 1991 Jun;173(11):3507–3513. doi: 10.1128/jb.173.11.3507-3513.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Massanari R. M., Pfaller M. A., Wakefield D. S., Hammons G. T., McNutt L. A., Woolson R. F., Helms C. M. Implications of acquired oxacillin resistance in the management and control of Staphylococcus aureus infections. J Infect Dis. 1988 Oct;158(4):702–709. doi: 10.1093/infdis/158.4.702. [DOI] [PubMed] [Google Scholar]
  21. Massidda O., Montanari M. P., Varaldo P. E. Evidence for a methicillin-hydrolysing beta-lactamase in Staphylococcus aureus strains with borderline susceptibility to this drug. FEMS Microbiol Lett. 1992 May 1;71(3):223–227. doi: 10.1016/0378-1097(92)90713-x. [DOI] [PubMed] [Google Scholar]
  22. McDougal L. K., Thornsberry C. The role of beta-lactamase in staphylococcal resistance to penicillinase-resistant penicillins and cephalosporins. J Clin Microbiol. 1986 May;23(5):832–839. doi: 10.1128/jcm.23.5.832-839.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Montanari M. P., Tonin E., Biavasco F., Varaldo P. E. Further characterization of borderline methicillin-resistant Staphylococcus aureus and analysis of penicillin-binding proteins. Antimicrob Agents Chemother. 1990 May;34(5):911–913. doi: 10.1128/aac.34.5.911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murakami K., Minamide W., Wada K., Nakamura E., Teraoka H., Watanabe S. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol. 1991 Oct;29(10):2240–2244. doi: 10.1128/jcm.29.10.2240-2244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murakami K., Nomura K., Doi M., Yoshida T. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1987 Sep;31(9):1307–1311. doi: 10.1128/aac.31.9.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Myers J. P., Linnemann C. C., Jr Bacteremia due to methicillin-resistant Staphylococcus aureus. J Infect Dis. 1982 Apr;145(4):532–536. doi: 10.1093/infdis/145.4.532. [DOI] [PubMed] [Google Scholar]
  27. Predari S. C., Ligozzi M., Fontana R. Genotypic identification of methicillin-resistant coagulase-negative staphylococci by polymerase chain reaction. Antimicrob Agents Chemother. 1991 Dec;35(12):2568–2573. doi: 10.1128/aac.35.12.2568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Relman D. A., Loutit J. S., Schmidt T. M., Falkow S., Tompkins L. S. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med. 1990 Dec 6;323(23):1573–1580. doi: 10.1056/NEJM199012063232301. [DOI] [PubMed] [Google Scholar]
  29. Ryffel C., Kayser F. H., Berger-Bächi B. Correlation between regulation of mecA transcription and expression of methicillin resistance in staphylococci. Antimicrob Agents Chemother. 1992 Jan;36(1):25–31. doi: 10.1128/aac.36.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rys P. N., Persing D. H. Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J Clin Microbiol. 1993 Sep;31(9):2356–2360. doi: 10.1128/jcm.31.9.2356-2360.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Saravolatz L. D., Markowitz N., Arking L., Pohlod D., Fisher E. Methicillin-resistant Staphylococcus aureus. Epidemiologic observations during a community-acquired outbreak. Ann Intern Med. 1982 Jan;96(1):11–16. doi: 10.7326/0003-4819-96-1-11. [DOI] [PubMed] [Google Scholar]
  32. Sierra-Madero J. G., Knapp C., Karaffa C., Washington J. A. Role of beta-lactamase and different testing conditions in oxacillin-borderline-susceptible staphylococci. Antimicrob Agents Chemother. 1988 Dec;32(12):1754–1757. doi: 10.1128/aac.32.12.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Song M. D., Wachi M., Doi M., Ishino F., Matsuhashi M. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 1987 Aug 31;221(1):167–171. doi: 10.1016/0014-5793(87)80373-3. [DOI] [PubMed] [Google Scholar]
  34. Tesch W., Strässle A., Berger-Bächi B., O'Hara D., Reynolds P., Kayser F. H. Cloning and expression of methicillin resistance from Staphylococcus epidermidis in Staphylococcus carnosus. Antimicrob Agents Chemother. 1988 Oct;32(10):1494–1499. doi: 10.1128/aac.32.10.1494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thornsberry C., Caruthers J. Q., Baker C. N. Effect of temperature on the in vitro susceptibility of Staphylococcus aureus to penicillinase-resistant penicillins. Antimicrob Agents Chemother. 1973 Sep;4(3):263–269. doi: 10.1128/aac.4.3.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thornsberry C., McDougal L. K. Successful use of broth microdilution in susceptibility tests for methicillin-resistant (heteroresistant) staphylococci. J Clin Microbiol. 1983 Nov;18(5):1084–1091. doi: 10.1128/jcm.18.5.1084-1091.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tokue Y., Shoji S., Satoh K., Watanabe A., Motomiya M. Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1992 Jan;36(1):6–9. doi: 10.1128/aac.36.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tomasz A., Drugeon H. B., de Lencastre H. M., Jabes D., McDougall L., Bille J. New mechanism for methicillin resistance in Staphylococcus aureus: clinical isolates that lack the PBP 2a gene and contain normal penicillin-binding proteins with modified penicillin-binding capacity. Antimicrob Agents Chemother. 1989 Nov;33(11):1869–1874. doi: 10.1128/aac.33.11.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ubukata K., Yamashita N., Konno M. Occurrence of a beta-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob Agents Chemother. 1985 May;27(5):851–857. doi: 10.1128/aac.27.5.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Utsui Y., Yokota T. Role of an altered penicillin-binding protein in methicillin- and cephem-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 1985 Sep;28(3):397–403. doi: 10.1128/aac.28.3.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wade J. C., Schimpff S. C., Newman K. A., Wiernik P. H. Staphylococcus epidermidis: an increasing cause of infection in patients with granulocytopenia. Ann Intern Med. 1982 Oct;97(4):503–508. doi: 10.7326/0003-4819-97-4-503. [DOI] [PubMed] [Google Scholar]
  42. Watanakunakorn C. Effect of inoculum size on in vitro susceptibility of methicillin-resistant Staphylococcus aureus to eighteen antimicrobial agents. Eur J Clin Microbiol. 1985 Feb;4(1):68–70. doi: 10.1007/BF02148668. [DOI] [PubMed] [Google Scholar]
  43. Watanakunakorn C. Treatment of infections due to methicillin-resistant Staphylococcus aureus. Ann Intern Med. 1982 Sep;97(3):376–378. doi: 10.7326/0003-4819-97-3-376. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES