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

Published online by Cambridge University Press:  02 January 2015

Thomas A. Wichelhaus ,
Klaus-Peter Hunfeld ,
Boris Böddinghaus ,
Peter Kraiczy ,
Volker Schàfer  and
Volker Brade
Thomas A. Wichelhaus*
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
Klaus-Peter Hunfeld
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
Boris Böddinghaus
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
Peter Kraiczy
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
Volker Schàfer
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
Volker Brade
Affiliation:
Institute of Medical Microbiology, University Hospital of Frankfurt, Frankfurt am Main, Germany
*
Institute of Medical Microbiology, University Hospital of Frankfurt, Paul-Ehrlich-Str. 40, 60596 Frankfurt am Main, Germany
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Abstract

Objective:

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

Design:

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.

Results:

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.

Conclusions:

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.

Type
Original Articles
Information
Infection Control & Hospital Epidemiology , Volume 22 , Issue 5 , May 2001 , pp. 294 - 298
Copyright
Copyright © The Society for Healthcare Epidemiology of America 2001

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References

1.Emori, TG, Gaynes, RP. An overview of nosocomial infections, including the role of the microbiology laboratory. Clin Microbiol Rev 1993;6:428–442.Google Scholar
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;274:639–644.CrossRefGoogle ScholarPubMed
3.Ayliffe, GAJ. The progressive intercontinental spread of methicillin-resistant Staphylococcus aureus. Clin Infect Dis 1997;24:S74–S79.Google Scholar
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;36:167–170.Google Scholar
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;5:9–17.CrossRefGoogle ScholarPubMed
6.Weber, S, Pfaller, MA, Herwaldt, LA. Role of molecular epidemiology in infection control. Infect Dis Clin North Am 1997;11:257–278.Google Scholar
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;18:426–439.Google Scholar
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;35:339–346.Google Scholar
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;33:551–555.CrossRefGoogle ScholarPubMed
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;35:1415–1423.CrossRefGoogle ScholarPubMed
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;36:1128–1134.Google Scholar
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;34:1502–1505.Google Scholar
13.Wichelhaus, TA, Kern, S, Schäfer, V, Brade, V. Rapid detection of epidemic strains of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 1999;37:690–693.CrossRefGoogle ScholarPubMed
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;33:2233–2239.CrossRefGoogle ScholarPubMed
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;173:7416–7422.Google Scholar
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;58:283–295.Google Scholar
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;102:1177–1186.Google Scholar
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;35:2580–2587.Google Scholar
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;32:846–847.Google Scholar
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;35:2514–2520.CrossRefGoogle ScholarPubMed
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;36:1083–1089.Google Scholar
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;35:881–885.Google Scholar
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;37:570–574.Google Scholar
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;32:305–317.Google Scholar
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;47:341–351.Google Scholar
26.Schwarzkopf, A, Karch, H. Genetic variation in Staphylococcus aureus coagulase genes: potential and limits for use as epidemiological marker. J Clin Microbiol 1994;32:2407–2412.CrossRefGoogle ScholarPubMed
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;121:507–514.Google Scholar
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;33:1537–1547.CrossRefGoogle Scholar
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;19:602–607.CrossRefGoogle ScholarPubMed
30.van Belkum, A. Amplification-based DNA fingerprinting: from artifactual to definitive typing and in between. J Clin Microbiol 1997;35:3008–3009.Google Scholar
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;126:29–35.Google Scholar
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;119:59–63.Google Scholar