title: 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.
abstract: 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.
authors.affiliation: Laboratory of Hospital Infection, Central Public Health Laboratory, London NW9 5HT, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Murchan
authors.forename: Mary Elizabeth
authors.initials: ME
authors.lastname: Kaufmann
authors.forename: Ariane
authors.initials: A
authors.lastname: Deplano
authors.forename: Raf
authors.initials: R
authors.lastname: de Ryck
authors.forename: Marc
authors.initials: M
authors.lastname: Struelens
authors.forename: Christina Elsberg
authors.initials: CE
authors.lastname: Zinn
authors.forename: Vivian
authors.initials: V
authors.lastname: Fussing
authors.forename: Saara
authors.initials: S
authors.lastname: Salmenlinna
authors.forename: Jaana
authors.initials: J
authors.lastname: Vuopio-Varkila
authors.forename: Névine
authors.initials: N
authors.lastname: El Solh
authors.forename: Christina
authors.initials: C
authors.lastname: Cuny
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Witte
authors.forename: Panayotis T
authors.initials: PT
authors.lastname: Tassios
authors.forename: Nikolas
authors.initials: N
authors.lastname: Legakis
authors.forename: Willem
authors.initials: W
authors.lastname: van Leeuwen
authors.forename: Alex
authors.initials: A
authors.lastname: van Belkum
authors.forename: Anna
authors.initials: A
authors.lastname: Vindel
authors.forename: Idoia
authors.initials: I
authors.lastname: Laconcha
authors.forename: Javier
authors.initials: J
authors.lastname: Garaizar
authors.forename: Saara
authors.initials: S
authors.lastname: Haeggman
authors.forename: Barbro
authors.initials: B
authors.lastname: Olsson-Liljequist
authors.forename: Ulrika
authors.initials: U
authors.lastname: Ransjo
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Coombes
authors.forename: Barry
authors.initials: B
authors.lastname: Cookson
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods* / Q000592:standards*; D004269:DNA, Bacterial / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods / Q000592:standards; D005062:European Union; D006801:Humans; D007753:Laboratories; D016106:Methicillin Resistance; D013203:Staphylococcal Infections / Q000453:epidemiology / Q000382:microbiology / Q000635:transmission; D013211:Staphylococcus aureus / Q000145:classification* / Q000187:drug effects* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific