Skip to main content Accessibility help

Rapid Molecular Typing of Methicillin-Resistant Staphylococcus aureus by PCR-RFLP

  • Thomas A. Wichelhaus (a1), Klaus-Peter Hunfeld (a1), Boris Böddinghaus (a1), Peter Kraiczy (a1), Volker Schàfer (a1) and Volker Brade (a1)...



To establish a new, rapid, and reliable genotypic fingerprinting technique for methicillin-resistant Staphylococcus aureus (MRSA) typing in routine epidemiological surveillance.


The method is based on polymerase chain reaction (PCR) restriction fragment-length polymorphism (RFLP) following HaeII digestion of simultaneously amplified parts of the protein A gene, the coagulase gene, and the hypervariable region adjacent to mecA. A total of 46 MRSA initial isolates were analyzed, including 14 isolates from five countries; the six German epidemic strains; 16 isolates from the Frankfurt metropolitan area, which were known to be heterogeneous by pulsed-field gel electrophoresis (PFGE); and 10 isolates obtained during three epidemics, all of which displayed an identical genotype.


Restriction analysis by PCR-RFLP permitted discrimination of 10 of 14 international isolates, all six German epidemic strains, and 15 of 16 national isolates. It also confirmed the homogeneous character of the 10 outbreak isolates.


This new and rapid PCR-RFLP typing method is an attractive tool in routine epidemiological surveillance. Its impressive characteristics are ease of performance and interpretation, while at the same time guaranteeing good discriminatory power, reproducibility, and typeability.


Corresponding author

Institute of Medical Microbiology, University Hospital of Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany


Hide All
1.Emori, TG, Gaynes, RP. An overview of nosocomial infections, including the role of the microbiology laboratory. Clin Microbiol Rev 1993;6:428442.
2.Vincent, JL, Bihari, DJ, Suter, PM, Bruining, HA, White, J, Nicolas-Chanoin, MH, et al. The prevalence of nosocomial infection in intensive care units in Europe. Results of the European Prevalence of Infection. JAMA 1995;274:639644.
3.Ayliffe, GAJ. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clin Infect Dis 1997;24:S74S79.
4.Humphreys, H, Duckworth, G. Methicillin-resistant Staphylococcus aureus (MRSA)—a re-appraisal of control measures in the light of changing circumstances. J Hosp Infect 1997;36:167170.
5.Rubin, RJ, Harrington, CA, Poon, A, Dietrich, K, Greene, JA, Moiduddin, A. The economic impact of Staphylococcus aureus infection in New York City hospitals. Emerg Infect Dis 1999;5:917.
6.Weber, S, Pfaller, MA, Herwaldt, LA. Role of molecular epidemiology in infection control. Infect Dis Clin North Am 1997;11:257278.
7.Tenover, FC, Arbeit, RD, Goering, RV. How to select and interpret molecular strain typing methods for epidemiological studies of bacterial infections: a review for healthcare epidemiologists. Infect Control Hosp Epidemiol 1997;18:426439.
8.Tyler, KD, Wang, G, Tyler, SD, Johnson, WM. Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J Clin Microbiol 1997;35:339346.
9.Bannerman, TL, Hancock, GA, Tenover, FC, Miller, JM. Pulsed-field gel electrophoresis as a replacement for bacteriophage typing of Staphylococcus aureus. J Clin Microbiol 1995;33:551555.
10.Morvan, A, Aubert, S, Godard, C, El Solh, N. Contribution of a typing method based on IS256 probing of SmaI-digested cellular DNA to discrimination of European phage type 77 methicillin-resistant Staphylococcus aureus strains. J Clin Microbiol 1997;35:14151423.
11.Olmos, A, Camarena, JJ, Nogueira, JM, Navarro, JC, Risen, J, Sánchez, R. Application of an optimized and highly discriminatory method based on arbitrarily primed PCR for epidemiologic analysis of methicillin-resistant Staphylococcus aureus nosocomial infections. J Clin Microbiol 1998;36:11281134.
12.Cuny, C, Witte, W. Typing of Staphylococcus aureus by PCR for DNA sequences flanked by transposon Tn916 target region and ribosomal binding site. J Clin Microbiol 1996;34:15021505.
13.Wichelhaus, TA, Kern, S, Schäfer, V, Brade, V. Rapid detection of epidemic strains of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 1999;37:690693.
14.Tenover, FC, Arbeit, RD, Goering, RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:22332239.
15.Ryffel, C, Bucher, R, Kayser, FH, Berger-Bachi, B. The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase. J Bacteriol 1991;173:74167422.
16.Shuttleworth, HL, Duggleby, CJ, Jones, SA, Atkinson, T, Minton, NP. Nucleotide sequence analysis of the gene for protein A from Staphylococcus aureus Cowan 1 (NCTC 8530) and its enhanced expression in Escherichia coli. Gene 1987;58:283295.
17.Kaida, S, Miyata, T, Yoshizawa, Y, Kawabata, S, Morita, T, Igarashi, H, et al. Nucleotide sequence of the staphylocoagulase gene: its unique COOH-terminal 8 tandem repeats. J Biochem (Tokyo) 1987;102:11771186.
18.Deplano, A, Vaneechoutte, M, Verschraegen, G, Struelens, MJ. Typing of Staphylococcus aureus and Staphylococcus epidermidis strains by PCR analysis of Inter-IS256 spacer length polymorphisms. J Clin Microbiol 1997;35:25802587.
19.Frénay, HM, Theelen, JP, Schouls, LM, Vandenbroucke-Grauls, CM, Verhoef, J, van Leeuwen, WJ, et al. Discrimination of epidemic and nonepidemic methicillin-resistant Staphylococcus aureus strains on the basis of protein A gene polymorphism. J Clin Microbiol 1994;32:846847.
20.Hoefnagels-Schuermans, A, Peetermans, WE, Struelens, MJ, van Lierde, S, van Eldere, J. Clonal analysis and identification of epidemic strains of methicillin-resistant Staphylococcus aureus by antibiotyping and determination of protein A gene and coagulase gene polymorphisms. J Clin Microbiol 1997;35:25142520.
21.Hookey, JV, Richardson, JF, Cookson, BD. Molecular typing of Staphylococcus aureus based on PCR restriction fragment polymorphism and DNA sequence analysis of the coagulase gene. J Clin Microbiol 1998;36:10831089.
22.Kumari, DNP, Keer, V, Hawkey, PM, Parnell, P, Joseph, N, Richardson, JF, et al. Comparison and application of ribosome spacer DNA amplicon polymorphisms and pulsed-field gel electrophoresis for differentiation of methicillin-resistant Staphylococcus aureus strains. J Clin Microbiol 1997;35:881885.
23.Marco, JY, Soriano, AC, Salazar, MS, Moral, CH, Ramos, SS, Smeltzer, MS, et al. Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene. J Clin Microbiol 1999;37:570574.
24.Nada, T, Ichiyama, S, Osada, Y, Ohta, M, Shimokata, K, Kato, N, et al. Comparison of DNA fingerprinting by PFGE and PCR-RFLP of the coagulase gene to distinguish MRSA isolates. J Hosp Infect 1996;32:305317.
25.Schmitz, FJ, Steiert, M, Tichy, HV, Hofmann, B, Verhoef, J, Heinz, HP, et al. Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods. J Med Microbiol 1998;47:341351.
26.Schwarzkopf, A, Karch, H. Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker. J Clin Microbiol 1994;32:24072412.
27.Walker, J, Borrow, R, Edwards-Jones, V, Oppenheim, BA, Fox, AJ. Epidemiological characterization of methicillin-resistant Staphylococcus aureus isolated in the North West of England by protein A (spa) and coagulase (coa) gene polymorphisms. Epidemiol Infect 1998;121:507514.
28.van Belkum, A, Khrytmans, J, van Leeuwen, W, Bax, R, Quint, W, Peters, E, et al. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J Clin Microbiol 1995;33:15371547.
29.van Belkum, A, Hermans, PW, Licciardello, L, Stefani, S, Grubb, W, van Leeuwen, W, et al. Polymerase chain reaction-mediated typing of microorganisms: tracking dissemination of genes and genomes. Electrophoresis 1998;19:602607.
30.van Belkum, A. Amplification-based DNA fingerprinting: from artifactual to definitive typing and in between. J Clin Microbiol 1997;35:30083009.
31.Nishi, J, Miyanohara, H, Nakajima, T, Kitajima, I, Yoshinaga, M, Maruyama, I, et al. Molecular typing of the methicillin resistance determinant (mec) of clinical strains of Staphylococcus based on mec hypervariable region length polymorphisms. J Lab Clin Med 1995;126:2935.
32.Sakurada, J, Li, Z, Seki, K, Murai, M, Usui, A, Morihigashi, M, et al. Biochemical and genetic heterogeneity of staphylococcal protein A. FEMS Microbiol Lett 1994;119:5963.

Rapid Molecular Typing of Methicillin-Resistant Staphylococcus aureus by PCR-RFLP

  • Thomas A. Wichelhaus (a1), Klaus-Peter Hunfeld (a1), Boris Böddinghaus (a1), Peter Kraiczy (a1), Volker Schàfer (a1) and Volker Brade (a1)...


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed