Methicillin-resistant Staphylococcus aureus (MRSA) is characterized by a large heterologous mobile genetic element—the staphylococcal chromosomal cassette, or SCCmec (7)—carrying the mecA gene, the central element of methicillin resistance, and the ccrAB locus, which encode recombinases involved in SCCmec mobility (11). SCCmec types are defined by combining the information on the genetic organization of the mec complex with the ccrAB allotype (6, 15). In S. aureus, three major mec complex classes have been described: class A contains the complete mec complex (mecI-mecR1-mecA), and classes B and C have the mecA regulatory genes disrupted due to the presence of insertion sequences (ΨIS1272-ΔmecR1-mecA and IS431-ΔmecR1-mecA, respectively) (8, 10). Concerning the ccrAB locus, three major allotypes (ccrAB1 to ccrAB3) (6) and one sporadic allotype (ccrAB4) (17) have been identified. Recently, a new type of ccr gene complex, which consists of only one gene (ccrC) not closely related to the ccrA or ccrB gene, was reported (8). SCCmec carries other sequences that define the overall genetic organization of the resistance cassette. These regions may be used as targets for typing strategies (1, 16, 22), and polymorphisms within these regions, particularly in the region downstream of the ccrAB genes (the J1 region), define SCCmec subtypes or variants (9, 12, 15, 17, 21).

After the description of SCCmec types I, II, and III (6), almost simultaneously two groups reported on the existence of a fourth structural type, named SCCmec type IV by both studies (12, 17). However, although those SCCmec elements are similar in size and share the same class of mec complex and the same genetic organization in the mecA downstream vicinity, they differ in the ccrAB allotype (ccrAB2 versus the previously unidentified ccrAB4) and in the J1 region. Later, SCCmec type IV with ccrAB allotype 2 was shown to be dominant among the emerging community-acquired MRSA strains, stressing its epidemiological relevance, whereas ccrAB allotype 4 remained extremely rare. Recently, ccrAB complexes closely related to ccrAB4 have been identified in a composite SCC structure in a Staphylococcus epidermidis strain (14) and in a MRSA clone belonging to clonal complex 45 and circulating in Zurich, Switzerland (18).

Strain HDE288 is the prototype strain for the SCCmec characterized by ccrAB4, and its SCCmec element has previously been fully characterized by PCR screenings and nucleotide sequencing (17). This strain is also the prototype of the so-called “pediatric” MRSA clone (sequence type 5), which was dominant in a pediatric hospital in Portugal in 1992 (19). The “pediatric” MRSA clone has also been detected in Poland, Argentina, Colombia, and the United States (4, 13, 19). In order to access the epidemiological relevance of this SCCmec element characterized by ccrAB4, we assembled a collection of MRSA strains belonging to the “pediatric” clone from several hospitals and cities in Portugal and also from international sources (Table ​(Table11).

All strains were initially typed by the SCCmec multiplex PCR strategy (16) and presumptively assigned to SCCmec type IV: class B for mec complex (mecI negative), mecA downstream vicinity typical of SCCmec types I, II, or IV (dcs positive), and mecA upstream vicinity negative for elements specific for SCCmec types I to III. Assignment to the class B mec complex (ΨIS1272-ΔmecR1-mecA) was confirmed for all strains by PCR with the following primers specific for ΨIS1272 and mecR1 (the location relative to the sequence of NCTC10442 [6], GenBank/EMBL/DDBJ accession number AB033763, is given in parentheses after the primer sequence): ISF1 (AAT TGA AGC AAA TGC CAA TCG) (positions 28812 to 28832) and MRP1 (CAA CTG TCG TAG TCG AAA CC) (positions 30734 to 30715). All strains were positive, with an amplicon similar in size to that of the prototype strain of SCCmec type I, characterized by a class B mec complex (6). ccrAB typing was then performed by PCR detection with primers specific for ccrAB allotypes 2 (15) and 4 (ccrAB4 F1 [TCA TCA ATA AGT ATG GAA CG] and ccrAB4 R1 [TTT CTT GCG ACT CTC TTG G]). Strains with ccrAB2 were classified as SCCmec type IV.

Strains positive for ccrAB allotype 4 were further characterized for the mecA upstream vicinity with primers designed based on the previously determined sequence of strain HDE288 (GenBank/EMBL/DDBJ accession number AF411935) (17). The primer sequences are as follows (with the location relative to the HDE288 sequence given in parentheses where appropriate): HDP9, CCC TCC AAA TTA TTA TCT CC (positions 54 to 73); HDP17, GCA ATT AGT TAC AAA GCA GC (positions 2833 to 2814); HDP18, CAT CTT CAA AGA CTT TTA GTC C (positions 2461 to 2482); HDP8, ACT AAC GGT AAA ACA TGA CC (positions 6925 to 6904); HDP2, TTA AAA GAT GCC AAC GAA GG (positions 9382 to 9401); ISR2, ATT CGT CGA ATT CAT TGT CAG G (specific for ΨIS1272 outwards). Primers HDP9 and HDP17 amplify within the J1 region close to the chromosomal junction; primers HDP18 and HDP8 amplify within the J1 region close to the ccrAB locus; and primers HDP2 and ISR2 amplify the region between the ccrAB locus and ΨIS1272. PCRs were performed in a T1 thermocycler (Biometra, Goettingen, Germany) under the following conditions: 94°C for 4 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s or 2 min; and a final extension at 72°C for 4 min. In each reaction (final volume, 50 μl), 5 ng of chromosomal template, 1.25 U of Amplitaq DNA polymerase (Applied Biosystems, Foster City, CA), and 20 pmol of each primer were used in 1× PCR buffer with MgCl₂ at 1.5 mM (Applied Biosystems) and a deoxynucleoside triphosphate mix at 0.16 mM (MBI Fermentas, Hanover, MD). All ccrAB4 strains gave positive signals with the expected size after PCR amplifications with the three primer pairs.

The SCCmec type characteristic of strain HDE288 was found in all 15 isolates from the same hospital (Hospital Dona Estefânia, Lisbon, Portugal) and in 4 other isolates from three hospitals, two located in the same city (Hospital Pulido Valente and Instituto Português de Oncologia, Lisbon, Portugal) and one located in a different city (Hospital da Universidade de Coimbra, Coimbra, Portugal). The remaining Portuguese isolates and all international isolates were characterized by ccrAB allotype 2. Since the initial assignment of strains to the pediatric MRSA clone was based on pulsed-field gel electrophoresis (PFGE) patterns, we have further characterized the genetic background of some strains by spa typing and multilocus sequence typing (MLST), as previously described (2, 3, 5, 20). All strains analyzed were characterized by sequence type 5 and by closely related spa types (TJMBDMGMK motif, type 311) (Table ​(Table11).

These results show that the SCCmec type previously found only in strain HDE288 is also present in other local strains belonging to the same clone, as defined by PFGE and confirmed by spa typing and MLST. Interestingly, most strains belonging to the pediatric MRSA clone are not characterized by this SCCmec type, suggesting at least two acquisitions of the mecA gene in the same genetic background. Still, the SCCmec type found in strain HDE288, defined by a class B mec complex, ccrAB allotype 4, and a specific J1 region, is epidemiologically relevant, and therefore we propose that it be identified as SCCmec type VI or type 4B, according to new SCCmec nomenclature proposed by Chongtrakool et al. (1).