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Antimicrob Agents Chemother. Jan 2007; 51(1): 390–393.
Published online Nov 6, 2006. doi:  10.1128/AAC.00921-06
PMCID: PMC1797639

Mosaic Staphylococcal Cassette Chromosome mec Containing Two Recombinase Loci and a New mec Complex, B2[down-pointing small open triangle]

Abstract

A novel staphylococcal cassette chromosome (SCC) mec from a clinical methicillin-resistant Staphylococcus aureus isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete ccr loci and a new variant of mec complex B, with ΔmecR1 interrupted by the aminoglycoside resistance transposon Tn4001.

Methicillin resistance in Staphylococcus aureus (MRSA) is facilitated by the acquisition of the staphylococcal cassette chromosome mec (SCCmec), which integrates site specifically into the staphylococcal genome and carries mecA, encoding the alternative penicillin-binding protein PBP2a, a β-lactam-insensitive transpeptidase (6, 11, 13, 22, 26). The precise excision and site- as well as orientation-specific integration of this element depend on the action of cassette chromosome recombinase genes (ccr's) located within the element (13).

Five main types of SCCmec have been described so far, each differing in size and composition and characterized according to its type of ccr locus and mec complex (3, 7, 25). mec complexes differ in the extents of insertion sequence (IS)-mediated deletions in the mecA regulatory genes mecR1 and mecI and the presence and location of insertion sequence IS431, IS1182, or IS1272 (23). Apart from the ccr and mec complexes, and some common mobile resistance elements, SCCmec subtypes harbor variable J (junkyard) regions containing truncated and nonessential genes and genes of unknown functions (8). In addition to the major types, a number of new SCC elements, including non-mecA-carrying cassettes, have recently been discovered (4, 5, 9, 14, 15, 17, 19).

An epidemiological study of methicillin-resistant staphylococci from Zurich in 2003 identified several strains which contained multiple ccr loci (21). Here, we describe the SCCmec of one of these isolates, MRSAZH47.

MRSAZH47 is of multilocus sequence type 100 and belongs to clonal complex 5, a genotype previously identified in Argentina (1, 24). In addition to its β-lactam resistance, it was resistant to aminoglycosides and carried a blaZ-encoded penicillinase. Its SCCmec type could not be determined by standard multiplex PCR (20), and additional ccr typing indicated that it contained both ccr2 and ccrC loci (21).

Southern hybridization of SmaI-digested chromosomal DNA, separated by pulsed-field gel electrophoresis, showed that mecA- and ccr2-hybridizing sequences were colocated on a separate SmaI fragment from the ccrC-hybridizing sequence (data not shown).

Transient overexpression of ccrAB2, facilitating the precise excision of all major SCCmec types (10, 12, 13, 18), was used to cure MRSAZH47. Southern hybridization showed that the resulting oxacillin-susceptible clone MRSAZH47c had lost mecA and both ccr loci, indicating that all three were present on a single excisable SCC element containing an internal SmaI restriction site (data not shown). The susceptibility profile of MRSAZH47c showed that aminoglycoside resistance had also been lost.

A cosmid library of MRSAZH47 DNA, consisting of over 600 clones with estimated inserts of about 45 kb, was constructed using a SuperCos1 cosmid vector kit (Stratagene, La Jolla, CA). Screening of the library by colony blot analysis using ccr2- or ccrC-specific probes identified 11 clones that hybridized to ccr2, 7 that hybridized to ccrC, and 2 that hybridized to both probes. Cosmids were end sequenced and the sequences compared to the genome sequence of S. aureus Mu50, revealing that the two cosmids hybridizing with both probes each contained one end of the SCCmec element and together completely covered it (data not shown).

Primers specific for known orfX, ccrC2, IS431, and mecA nucleotide sequences were used to synthesize long-range PCR products that were subcloned into either pUC19 or pBluescript SK(+). Inserts were end sequenced and the obtained sequences assembled. The double-stranded nucleotide sequence of the 33.7-kb element was completed by primer walking.

This SCCmec proved to be unique, containing elements and properties not previously described. GeneMark.hmm (16) and BLASTX (2) identified 33 open reading frames (ORFs), all of which were identical or highly similar in sequence to previously annotated staphylococcal genes (Table (Table11 and Fig. Fig.11).

FIG. 1.
Genetic map of the mosaic SCCmec element from MRSAZH47. Open reading frames are symbolized by arrows, and the bars below them indicate homologies to previously described SCC elements. Integration site sequences for SCCmec (*), direct repeats of ...
TABLE 1.
ORFs encoded on SCCmecZH47

The orfX insertion site and the characteristic terminal inverted and direct repeats, generated upon insertion, were almost identical to those of other, previously described SCCmec elements (12). However, the left-end (proximal) direct repeat sequence contained a nucleotide transition of an adenine to guanine (Table (Table1),1), which has not been found elsewhere and increased the identity between the junctional direct repeats. This mutation did not impede the excision of the element, as demonstrated by the precise curing of SCCmecZH47 from the chromosome of MRSAZH47. It could, however, possibly influence the stability or transfer frequency of the element.

SCCmecZH47 contained a new mec complex that we have named B2 because of its similarity to mec complex B. The new B2 complex differed in that the 987-bp ΔmecR1 fragment was interrupted by insertion of the aminoglycoside resistance transposon Tn4001 at base pair position 820 (Fig. (Fig.1),1), and while the mecA promoter region was identical to that of mec complex B, the mecA gene sequence was identical to that of SCCmec types V and VT.

In addition to a ccrAB2 locus at the usual position downstream of mecA, the element possessed a ccrC locus between orfX and the dru element (Fig. (Fig.1).1). The ccrA2 sequence was identical to that of SCCmec type IVe, while the ccrB2 sequence was identical to that of SCCmec type IVd. Comparison of the ccrC sequence to published variants revealed high levels of similarity to the ccrC2 and ccrC3 sequences of SCCmec types VT and III, respectively (Table (Table11).

The element as a whole appeared mosaic in structure. The presence of both a ccrAB2 locus and a variant ccrC locus and of regions with strong similarity to several different SCC elements, including the typical hospital-acquired MRSA type III SCCmec, the community-associated MRSA SCCmec types IV and VT, and SCCmec from the non-S. aureus species Staphylococcus saprophyticus, suggests that SCCmecZH47 had been assembled via several recombination events (Fig. (Fig.11).

Most of the new SCC and SCCmec elements recently discovered, including the SCCmecZH47 described here, appear to have acquired regions from other SCC elements, suggesting that significant intra- and interspecies exchange and recombination of SCC DNA occurs.

Nucleotide sequence accession number.

The nucleotide sequence newly determined in this study was deposited in the EMBL database under accession number AM292304.

Acknowledgments

This study was supported by Swiss National Science Foundation grant NF31-105390/1.

Footnotes

[down-pointing small open triangle]Published ahead of print on 6 November 2006.

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