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-field gel 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,

Genotypic relatedness and characterization of Staphylococcus pseudintermedius associated with post-operative surgical infections in dogs

“…The PFGE was performed as described previously, using the following running conditions: 6 V cm 21 at 14 uC, with an initial switch time of 2 s and a final switch time of 5 s for 23 h (Murchan et al, 2003;Perreten et al, 2010). The macrorestriction profile was analysed using BioNumerics software version 5.10 (Applied Maths).…”

Recognition of SCC mec Types According to Typing Pattern Determined by Multienzyme Multiplex PCR-Amplified Fragment Length Polymorphism Analysis of Methicillin-Resistant Staphylococcus aureus

“…PFGE is the gold standard in typing of S. aureus, and a web-based database has been recently set up to compare intercenter typing patterns that allow tracing of isolates (10). However, for typing of large strain collections this method is less suitable since it is rather time-consuming and tedious.…”

“…Due to the policy of "search and destroy," MRSA is not endemic in The Netherlands (24). Pulsed-field gel electrophoresis (PFGE) is regarded as the gold standard for epidemiological typing of MRSA, and a new harmonized protocol for PFGE has been developed enabling the establishment of a European web-based database for comparison of intercenter MRSA PFGE patterns (10). However, PFGE is a time-consuming and tedious method and therefore is not best suited for use in the hospital clinical microbiology laboratory and in large-scale studies of outbreaks.…”

Methicillin-Resistant Staphylococcus aureus Infections in Uppsala County, Sweden

“…This prospective surveillance study of CA-MRSA and HCA-MRSA isolates was conducted from [2004][2005][2006] [16]. Staphylococcus protein A gene (spa) typing, a DNA sequence-based method for molecular analysis, was done as previously described [17].…”