Multiplex PCR Strategy for Rapid Identification of Structural Types and Variants of the mec Element in Methicillin-Resistant Staphylococcus aureus

Abstract

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...

Emergence of SCCmec Type IV as the Most Common Type of Methicillin-Resistant Staphylococcus aureus in a University Hospital

“…The SCCmec multiplex PCR assay was performed according to the method of Oliveira and de Lancastre [17] with modifications in the primer concentrations. Concentrations for primers were as follows: 0.25 lM for CIF2 F2, CIF2 R2, MECI P2, MECI P3, RIF5 F10, RIF5 R13; 0.3 lM for IS431 P4, pUB110 R1; 0.5 lM for pT181 R1; 1.0 lM for DCS F2, DCS R1, RIF4 F3, RIF4 R9, MECA P4, MECA P7 and 1.7 lM for KDP F1, KDP R1.…”

“…Concentrations for primers were as follows: 0.25 lM for CIF2 F2, CIF2 R2, MECI P2, MECI P3, RIF5 F10, RIF5 R13; 0.3 lM for IS431 P4, pUB110 R1; 0.5 lM for pT181 R1; 1.0 lM for DCS F2, DCS R1, RIF4 F3, RIF4 R9, MECA P4, MECA P7 and 1.7 lM for KDP F1, KDP R1. The multiplex PCR included eight loci (A-H) [17]. MRSA strains NCTC10442, N315, 85/2082, and WSPP A were included as controls for SCCmec types I, II, III, and IV, respectively.…”

Evolution of Sporadic Isolates of Methicillin-Resistant Staphylococcus aureus (MRSA) in Hospitals and Their Similarities to Isolates of Community-Acquired MRSA

“…Isolate HSA10 had ccr type 3 and mec complex type A and was therefore classified as SCCmec type III (19). However, by the multiplex PCR strategy it was determined that there was amplification of the locus corresponding to the dcs region that was not detectable in the SCCmec type III of previously characterized MRSA strains (19,28,29). Isolates JP87 and JP238 had ccr type 2 and mec complex type A and were therefore classified as SCCmec type II (19).…”

“…types I, II, III, and IV were associated with 6, 2, 2, and 11 different STs, respectively (Table 2). For four isolates (HSA10, JP87, JP238, and TAW166), the SCCmec type could not be directly inferred by the multiplex PCR strategy since the amplification patterns observed did not exactly correspond to the ones described by Oliveira et al (28). For these four isolates, SCCmec types were determined by PCR typing the mec gene and the ccr gene complexes (26).…”

“…The SCCmec types were determined by a multiplex PCR strategy (28). For all isolates classified as SCCmec type IV and whenever at least one of the structural features shown to be typical of a particular SCCmec type was not identified by this methodology, we used the PCR amplification of the ccr (for cassette chromosome recombinase) gene and the mec gene complex (26).…”