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Antimicrobial Agents and Chemotherapy, December 2005, p. 5129-5132, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5129-5132.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Type V Staphylococcal Cassette Chromosome mec in Community Staphylococci from Australia

F. G. O'Brien,1* G. W. Coombs,1,2 J. C. Pearson,2 K. J. Christiansen,1,2 and W. B. Grubb1

Gram-Positive Bacteria Typing and Research Unit, Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology,1 Department of Microbiology and Infectious Diseases, Royal Perth Hospital, Perth, Western Australia, Australia2

Received 12 May 2005/ Returned for modification 11 August 2005/ Accepted 8 September 2005


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ABSTRACT
 
Twenty Australian community staphylococci harboring the type V staphylococcal cassette chromosome mec (SCCmec) were found to belong to eight multilocus sequence types. Five were previously unreported novel type V SCCmec elements. The mec complexes were of two types, based on the polymorphisms in the IS431 transposase genes. Five isolates were multiresistant.


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TEXT
 
Community methicillin-resistant Staphylococcus aureus (CMRSA) isolates are emerging as significant pathogens in communities. Unlike the health care-associated methicillin-resistant S. aureus (HMRSA) isolates, CMRSA isolates are typically resistant to few non-beta-lactam antibiotics. Resistance to methicillin is mediated by the mec complex, which is encoded on a genomic resistance island called the staphylococcal cassette chromosome mec (SCCmec) (6). SCCmec is the genetic element most responsible for stable multiple-drug resistance in HMRSA and is characterized according to the class of mec complex and the type of cassette chromosome recombinases (ccr) that it harbors. Five types of SCCmec have been characterized (4, 6). Types I, II, and III are harbored by HMRSA isolates, while type IV occurs mostly in CMRSA isolates (11). In Australia, however, CMRSA isolates also harbor type V SCCmec, which encodes the class C2 mec complex, and the type 5 ccr gene, ccrC (4). In class C2 mec complex isolates, the mecA regulatory genes mecI and most of mecR1 are deleted by the insertion sequence IS431. This type of deletion was first described in clinical isolates of the coagulase-negative staphylococci (CoNS) Staphylococcus haemolyticus and Staphylococcus epidermidis (5). This study has investigated CMRSA and community CoNS isolates from Australia that harbor SCCmec elements that encode either the class C2 mec complex and ccrC (type V) or unusual combinations involving these elements (novel type V).

Twenty staphylococci (17 methicillin-resistant S. aureus [MRSA] isolates, 2 S. haemolyticus isolates [WBG10504 and WBG10505], and 1 S. epidermidis isolate [WBG10506]) isolated from different people in four states of Australia (Western Australia [WA], South Australia, Victoria, and Queensland) were investigated (Table 1). Two of the MRSA isolates (WBG8318 and WBG8404) and the three CoNS isolates were isolated from people in remote communities in WA in 1995. Isolates WCH100, IMVS67, RPH74, and RBH87 were obtained during a national multicenter surveillance study conducted in 2000. The remaining isolates were from WA and included a 2004 isolate from a nasal screening swab (isolate 04-17116) and isolates from clinical samples collected during 2003 and 2004 either from people in the community or from hospital patients who had been in hospital for less than 48 h.


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  		TABLE 1. Phenotypic and genotypic properties of the community methicillin-resistant staphylococci investigated in this studya

Antimicrobial susceptibility testing was performed according to the CLSI (formerly the NCCLS) recommendations (8). Multiresistance was classified according to previously published guidelines (12). Oxacillin MICs were determined by Etest (AB Biodisk, Solna, Sweden). The mecA gene was detected by using the primers mecAup (5'-TGCTATCCACCCTCAAACAGG-3') and mecAdn (5'-AACGTTGTAACCACCCCAAG-3') (5). Multilocus sequence typing (MLST) (2) and contour-clamped homogeneous electric field (CHEF) electrophoresis were performed as described previously (10). The SCCmec elements were typed by PCR; for the class C2 mec complex, the primers were mA (7) and IS2 (7); and for ccrC, the primers were {gamma}F and {gamma}R (4). The Panton Valentine leukocidin (PVL) determinant was detected by PCR (3), and its presence was confirmed by sequencing.

The results are summarized in Table 1. All the staphylococci contained mecA and, except for S. haemolyticus WBG10504, expressed oxacillin resistance. The MICs for oxacillin ranged from 0.25 mg/liter to >256 mg/liter. Five of the CMRSA isolates (03-16672, 03-17796, 04-17451, 04-16419, and 04-17116) were multiply resistant (12) (Table 1).

The PVL determinant was present only in the three clonally related sequence type 59 (ST59) isolates.

Sixteen staphylococci harbored SCCmec type V, and there were four novel type V SCCmec elements. Isolates 04-17489, RBH87, WBG10198 and WBG8404, harbored the type 5 ccrC determinant with mec complexes of classes B, B, E, and B1 (7), respectively, and 04-16419 encoded a class C2 mec complex with a type 2 ccr. This suggests that genetic recombination involving the mec complex has occurred in these CMRSA isolates.

The type V elements were found in eight unrelated MLST genetic lineages, two unrelated strains of S. haemolyticus (CHEF pattern similarity of 59%), and one S. epidermidis strain. This widespread dissemination of the type V elements across genetic backgrounds and species barriers supports the concept that they are mobile (13). ST5, ST8, ST129, ST30, and ST45 are well-established lineages of CMRSA in Australia, in which the majority of isolates except for ST45 harbor type IV SCCmec elements (1, 9). The presence of SCCmec type V elements in these lineages suggests that acquisition and recombination events are involved in the emergence of CMRSA in Australia.

The C2 mec complex was amplified with primers for the 5' terminus of mecA and the left-hand end of the IS431 that has deleted mecI and truncated mecR1. The resulting amplicons were sequenced. In each of these, mecR1 was truncated by IS431 at nucleotide (nt) 93. One nucleotide downstream of the mecR1 truncation site was a 13-bp imperfect inverted repeat (IR) sequence of nt 94-CaAAATaTTATGT{cjs0822}c{cjs0822}ACATAAgATTTgG-nt 120 (lowercase nucleotides are noncomplementary). This is 1 bp downstream from a previously reported natural truncation site found in class C2 mec complexes (5). The close proximity of these sites to the IR suggests that the structure may provide a site where IS431 truncation of mecR1 is more likely to occur.

There were 10-nt polymorphisms in the transposase gene (tnp) of the IS431 element truncating the C2 mec complexes (Fig. 1). This differentiated the isolates with type V SCCmec elements into two subgroups. The ST45 CMRSA isolates were in subgroup 1, with the remainder belonging to subgroup 2 (GenBank accession number DQ171730). Polymorphisms at nucleotide positions 144, 150, and 400 of the subgroup 2 tnp sequences have created three in-frame stop codons of TAG, TGA, and TAG, respectively. These polymorphisms would almost certainly stabilize the mec complex by ensuring that an active transposase is not produced. The more widespread distribution of SCCmec type V, subgroup 2 (five lineages of CMRSA isolates and three unrelated CoNS isolates), supports this hypothesis and also suggests that the subtype is more readily disseminated.



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  		FIG. 1. ClustalW alignment of the 5' 481 nucleotides of the truncating IS431 transposase gene. Nucleotide positions are indicated at the ends. Identical nucleotides are indicated by an asterisk; nonidentical nucleotides are in boldface and are indicated by a gap. Stop codons are boxed.

This study has shown not only that there is diversity among the community staphylococci that harbor the SCCmec type V and novel type V elements but also that there is structural and genetic diversity within the elements themselves. It is of concern that some of the CMRSA isolates are now multiply resistant. With CMRSA threatening to become a substantial health problem in the general community, where it will be very difficult to control its dissemination, it is now important that health authorities develop surveillance and antibiotic prescribing policies that will restrict both the spread of CMRSA isolates in the community and their acquisition of further resistance determinants.

Nucleotide sequence accession number. The partial sequence of the nonfunctional IS431 transposase was submitted to GenBank and assigned accession number DQ171730.


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ACKNOWLEDGMENTS
 
We gratefully acknowledge the following: the WA Genome Resource Centre, Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, Perth, WA, for sequencing; Cheryl McCullough from the Royal Perth Hospital Department of Microbiology and David Atlas, Rebecca Lee, Ngan Pham, and Mary Malkowski from the Gram-Positive Bacteria Typing and Research Unit for technical assistance; the Australian Group for Antimicrobial Resistance for CMRSA isolates; the Department of Health of WA for funding; and the National Health and Medical Research Council of Australia for a grant to W.B.G. for part of F.G.O.'s salary.


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FOOTNOTES
 
* Corresponding author. Mailing address: Curtin University of Technology, GPO Box U1987 Perth, Western Australia 6845, Australia. Phone: 61 8 92240302. Fax: 61 8 92240303. E-mail: F.G.O'Brien{at}curtin.edu.au. Back


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Antimicrobial Agents and Chemotherapy, December 2005, p. 5129-5132, Vol. 49, No. 12
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.12.5129-5132.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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