Full characterization of methicillin-resistant Staphylococcus aureus (MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous mec element these bacteria carry, which is associated with drug resistance determinant mecA. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the mec element types based on the structural features that were shown to be typical of mec elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated mec elements was previously determined by hybridization and PCR screenings and also by DNA sequencing. The method was tested together with multilocus sequence typing and other typing methods for the characterization of 18 isolates representative of the MRSA clones recovered during a hospital outbreak in Barcelona, Spain. The multiplex PCR was shown to be rapid, robust, and capable in a single assay of identifying five structural types of the mec element among these strains, three major and two minor variants, each one of which has been already been seen among MRSA characterized earlier. This technique should be a useful addition to the armamentarium of molecular typing tools for the characterization of MRSA clonal types and for the rapid tentative identification of structural variants of the mec element.Molecular typing techniques have been used with increasing frequency in studies of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) and also for a better understanding of the evolutionary relationships among MRSA clones (3,7,9,26). One of the conclusions emerging from these studies was that a complete characterization of MRSA lineages requires not only identification of the genetic background of the bacteria but also identification of the structural types of the large and heterologous mec element, which carries methicillin resistance determinant mecA (11,26,27).Studies by Ito et al. have elucidated the complete structure of three major mec elements, also referred to as the staphylococcal chromosomal cassette (SCCmec). Type I (34 kb) was identified in the first MRSA strain isolated in 1961 in the United Kingdom (strain NCTC10442), type II (52 kb) was identified in an MRSA strain isolated in 1982 in Japan (strain N315), and type III (66 kb) was identified in an MRSA strain isolated in 1985 in New Zealand (strain 82/2082) (12, 13). More recently, a smaller fourth mec element, SCCmec type IV (20 to 24 kb), was independently identified among representatives of the Pediatric clone (26) and in two community-acquired MRSA strains (18).In a recent report we described the characterization of the structural types of the mec elements carried by 28 MRSA isolates belonging to five widespread epidemic clones, which were all characterized by multilocus sequence typing (MLST) as single-locus or double-locus variants of two completel...

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Previous surveillance studies carried out by our laboratories, primarily in Southern and Eastern Europe, Latin America, and the United States, have characterized 3,067 methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates by a combination of molecular typing methods. Nearly 70% of these isolates could be classified into five clonal types showing extensive geographic spread. Representative isolates of these clonal types were now reexamined for their genetic relatedness by multilocus sequence typing (MLST) and by sequencing the polymorphic region of protein A (spaA typing), and also for the type of the Staphylococcal Chromosomal Cassette (SCCmec) resident in the bacteria. Three of the previously classified clonal types (Iberian, Brazilian, and Hungarian clones) shared a common or closely related genetic background A, which was the same as the background of the earliest European isolates of MRSA from England and Denmark. The Pediatric and New York/Japan clones belonged to a completely different genetic background B. The three recently described SCCmec types were specifically associated with different pandemic clones: types I and III with isolates of genetic background A and type II with isolates of genetic background B. A novel SCCmec related to type I, called SCCmec type IV, was identified in some MRSA strains belonging to genetic background A as well as B. Structural variations in SCCmec types I and III were also observed. The data allow tentative identification of an evolutionary pathway for the emergence of pandemic MRSA clones and also provide evidence for the multiple, yet restricted, numbers of acquisition of the mec element by S. aureus.

Staphylococcal cassette chromosome mec (SCCmec) typing is important for the identification and definition of methicillin-resistant Staphylococcus aureus clones, and for routine purposes, multiplex PCR assays are the most adequate for SCCmec typing. Here, we describe an update to the multiplex PCR strategy for SCCmec typing that we described in 2002 so that SCCmec types IV and V may be properly identified.