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.

FIG. 1.

Gel image obtained in center 1 following PFGE of representative type strains of European EMRSA by using the initial harmonized protocol and NCTC 8325 as the reference standard. Molecular sizes in kilobases are shown for the reference standard. Lanes 1, 6, 12, 18, 23, and 28, NCTC 8325; lane 2, Belgium E1; lane 3, Belgium E2; lane 4, Belgium E3; lane 5, lambda; lane 7, Finland E1; lane 8, Finland E5; lane 9, Finland E24; lane 10, Finland E10; lane 11, Finland E7; lane 13, France A; lane 14, France B; lane 15, France C; lane 16, Greece 1; lane 17, Spain E1; Lane 19, North German I; lane 20, South German II; lane 21, Hannover III; lane 22, Berlin IV; lane 24, United Kingdom EMRSA-1; lane 25, United Kingdom EMRSA-3; lane 26, United Kingdom EMRSA-15; lane 27, United Kingdom EMRSA-16.

FIG. 2.

Four examples of gel images of the 21 representative type strains of European EMRSA strains digested with SmaI and with the final harmonized protocol for PFGE as performed in 9 of the 10 laboratories. NCTC 8325 is the reference standard used to normalize the gels. Bands larger than 674 kb (as bands above the highest band of the standard will not be normalized) and smaller than 36 kb (the third-lowest band of the standard) (as resolution of bands below this is often poor) were not defined for the purpose of further analysis of the gel. (A) Example of a good gel. (B) An underrunning gel due to a lower running temperature. (C) An overloaded gel. (D) An inadequately destained gel. See the text for further details and the legend to Fig. 1 for the running order of the 21 strains.

FIG. 3.

Dendrogram of all 21 type strains from gels from seven laboratories analyzed in the Laboratory of Hospital Infection, Central Public Health Laboratory, using GelCompar with percent similarity calculated by Dice coefficient (tolerance of 1% and optimization of 0.5%) and represented by UPGMA. Bands larger than 674 kb (as bands above the highest band of the standard will not be normalized) and smaller than 36 kb (the third-lowest band of the standard), as resolution of bands below this is often poor, were not defined for the purpose of further analysis of the gel. Clones A, B, and C are shown (see text for details).

FIG. 3.

Dendrogram of all 21 type strains from gels from seven laboratories analyzed in the Laboratory of Hospital Infection, Central Public Health Laboratory, using GelCompar with percent similarity calculated by Dice coefficient (tolerance of 1% and optimization of 0.5%) and represented by UPGMA. Bands larger than 674 kb (as bands above the highest band of the standard will not be normalized) and smaller than 36 kb (the third-lowest band of the standard), as resolution of bands below this is often poor, were not defined for the purpose of further analysis of the gel. Clones A, B, and C are shown (see text for details).