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Journal of Clinical Microbiology, April 2003, p. 1574-1585, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1574-1585.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Harmonization of Pulsed-Field Gel Electrophoresis Protocols for Epidemiological Typing of Strains of Methicillin-Resistant Staphylococcus aureus: a Single Approach Developed by Consensus in 10 European Laboratories and Its Application for Tracing the Spread of Related Strains

Stephen Murchan,1 Mary Elizabeth Kaufmann,1 Ariane Deplano,2 Raf de Ryck,2 Marc Struelens,2 Christina Elsberg Zinn,3 Vivian Fussing,3 Saara Salmenlinna,4 Jaana Vuopio-Varkila,4 Névine El Solh,5 Christina Cuny,6 Wolfgang Witte,6 Panayotis T. Tassios,7 Nikolas Legakis,7 Willem van Leeuwen,8 Alex van Belkum,8 Anna Vindel,9 Idoia Laconcha,10 Javier Garaizar,10 Saara Haeggman,11 Barbro Olsson-Liljequist,11 Ulrika Ransjo,12 Geoffrey Coombes,13 and Barry Cookson1*

Laboratory of Hospital Infection, Central Public Health Laboratory, London NW9 5HT, United Kingdom,1 Laboratoire de Microbiologie, Hopital de Erasme, B-1070 Brussels, Belgium,2 Statens Serum Institut, DK-2300 Copenhagen-S, Denmark,3 National Public Health Institute, 00300 Helsinki, Finland,4 Unité des Staphylocoques, Insitut Pasteur, 75724 Paris Cedex 15, France,5 Bereich Wernigerode, Robert Koch-Institut, 38855 Wernigerode (Harz), Germany,6 Department of Microbiology, Medical School, University of Athens, 115 27 Athens, Greece,7 AZR Bacteriologie, Department of Medical MicrobiologyInfectious Diseases, Erasmus University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands,8 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid,9 Department of Immunology, Microbiology, and Parasitology, UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain,10 Swedish Institute for Infectious Disease Control, SE-171 82 Solna Division of Clinical Microbiology, Department of Laboratory Medicine Karolinska,11 Hospital, S-171 76 Stockholm, Sweden,12 Royal Perth Hospital, Perth, Western Australia, Australia,13

Received 19 August 2002/ Returned for modification 10 October 2002/ Accepted 18 December 2002

Pulsed-fieldgel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium, Finland, France, Germany, and Spain (and from the wider HARMONY collection in Portugal, Slovenia, and Sweden). Strains from the United Kingdom (EMRSA-15 and -16) have been identified in several othercountries, and other clonally related strains have also been identified. This highlights the need for closer international collaboration to monitor the spread of current epidemic strains as well as the emergence of new ones.

* Corresponding author. Mailing address: Laboratory of Hospital Infection, Central Public Health Laboratory, 61 Colindale Ave., London NW9 5HT, United Kingdom. Phone: 020 8200 4400. Fax: 020 8200 7449. E-mail: bcookson{at}phls.org.uk.

Journal of Clinical Microbiology, April 2003, p. 1574-1585, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1574-1585.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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