A convenience sample, totaling 2,358 MRSA isolates from human specimens, was submitted to the National Microbiology Laboratory (NML) for spa typing, as described (4,5). During January 2007–October 2008, the Saskatchewan Disease Control Laboratory submitted 2,008 specimens; during October 2007–August 2008, the Cadham Provincial Laboratory in Manitoba submitted 350 specimens. An additional 1,329 isolates from human specimens were spa typed by the Saskatchewan Disease Control Laboratory.

Given the client base of the Cadham Provincial Laboratory, most of these isolates would have originated from colonized and infected persons living in the community or in personal-care homes or from persons hospitalized in smaller community hospitals, whereas, for surveillance purposes, the Saskatchewan Disease Control Laboratory receives isolates from all colonized and infected persons across the province. Detailed information regarding why cultures were taken (e.g., screening admissions, outbreak investigations) and other clinical and epidemiologic data were limited. Isolates typed in this study represented ≈17% of all MRSA isolates from persons in Manitoba and ≈66% of all MRSA isolates from persons in Saskatchewan within the study period. An additional isolate was sent to the NML from Sunnybrook Health Sciences Centre in Ontario for reference purposes.

Isolates with spa types associated with ST398 were confirmed by multilocus sequence typing; tested for Panton-Valentine leukocidin toxin, mecA, and nuc genes; and typed for SCCmec as described (6–9). Pulsed-field gel electrophoresis (PFGE) of SmaI- or Cfr91-digested genomic DNA was conducted as described (10). Antimicrobial drug susceptibility testing was conducted by using standard broth microdilution panels according to Clinical and Laboratory Standards Institute guidelines (11). Breakpoints for fusidic acid and mupirocin resistance, which were not provided in the guidelines, were as described (12,13).

A fosmid library was constructed by cloning sheared genomic DNA from S. aureus isolate 08 BA 02176 into the pCC2FOS vector. The fosmid clones were screened by PCR to identify specific genes orfX, mecA, and the chromosomal region located downstream of SCCmec. Fosmid clone 1G1 was identified and contained the entire SCCmec region of the 08 BA 02176 isolate. Colonies were prepared by using the CopyControl Fosmid Library Production Kit (Epicenter Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. Fosmid DNA was column purified by using a QIAGEN Plasmid Mini Kit (QIAGEN, Valencia, CA, USA).

DNA sequencing was performed on the ABI3730xl genetic analyzer (Applied Biosystems, Foster City, CA, USA). Staden (Pregap4) software was used to prepare trace data for sequence assembly (14). Sequencing reads were assembled by using the Staden Gap4 program. Gap closure was achieved by primer walking and long-range PCR. Specific primers were designed near the ends of neighboring contigs (contiguous sequence of DNA created by overlapping sequenced fragments of a chromosome), and PCRs were performed with chromosomal template DNA. Regions containing putative frameshifts and point mutations were resequenced to verify the fidelity of the sequence.

Annotation and data mining of the S. aureus 08 BA 02176 1G1 fosmid clone sequence were performed by using the GenDB version 2.2 annotation tool (15). Putative protein coding sequences were determined according to coding sequence predictions of Glimmer, which is integrated into the GenDB package. Similarity searches were performed by using BLASTN and BLAST2P (www.ncbi.nlm.nih.gov/blast/Blast.cgi) against the nonredundant nucleotide and protein databases, respectively. Additionally, a BLAST2P search was performed against the databases nr (ftp://ftp.ncbi.nlm.nih.gov/blast/db/), SWISS-PROT (www.expasy.ch/sprot/), and KEGG-Genes (ftp://ftp.genome.jp/pub/kegg/genes/); the protein family databases Pfam (http://pfam.sanger.ac.uk/) and TIGRFAM (www.jcvi.org/cms/research/projects/tigrfams/overview/); and predictive signal peptide (Signal P [www.cbs.dtu.dk/services/SignalP/]) and transmembrane helix analysis (TMHMM [www.cbs.dtu.dk/services/TMHMM/]), the nonredundant database on protein level. An automatic functional annotation was followed by a manual annotation of each predicted gene.

The high prevalence of LA-MRSA colonization of pigs and pig farmers in Canada (2) and the United States (3) and this report of human infections suggest that this LA-MRSA strain from Canada poses potential public and occupational health concern in North America. This strain has been associated with various types of infections in pigs (17,18) and humans (19,20) and is transmissible from animal patients to veterinary workers (21), healthcare workers (22), and family members (1). Evidence also suggests that this strain might be spreading from animals to the environment, which may facilitate the colonization or infection of persons who are not involved in animal husbandry (23). Whereas in 2006 in the Netherlands LA-MRSA accounted for >20% of all MRSA isolated (24), carriage of this strain in the general population of 2 provinces in Canada (Manitoba and Saskatchewan) appears rare (0.14%). This difference could be attributed to the substantially higher density of pigs in the Netherlands (1,244 pigs/km²) than in Manitoba (55 pigs/km²), Saskatchewan (6 pigs/km²), and Ontario (91 pigs/km²) (www.agriculture.gov.sk.ca/Pig_Densities). It is also plausible that the much lower proportions of LA-MRSA in Canada, relative to a country with low MRSA endemicity such as the Netherlands, is attributable to competition with other highly successful human epidemic MRSA clones circulating in Canada, including CMRSA2 (USA200/800), CMRSA7 (USA400), and CMRSA10 (USA300) (25,26).

The tested LA-MRSA isolates were highly susceptible to most classes of antimicrobial drugs, except β-lactams and tetracyclines, the latter of which has been attributed to its high usage in animal husbandry (27). The complete sequence of the SCCmec region showed a novel SCCmecV subtype sharing sequence identity in its J1 and J3 regions with chromosomal genes in the S. epidermidis RP62A chromosome (GenBank accession no. CP000029), including a CRISPR system. CRISPRs and associated cas genes are present in many other bacterial (≈40%) and archaeal (≈90%) genomes (28,29) and have been shown to be involved in sequence-directed immunity against phages (30,31) and plasmids (32). The resistance to plasmids and phages encoded by this system could explain why many of these ST398-MRSA-V strains contain fewer antimicrobial drug resistance genes and phage-encoded virulence factors than do other epidemic MRSA strains (33,34). The origin of this CRISPR system is unknown, but the propagation of CRISPR loci throughout prokaryote genomes has been proposed to occur through horizontal gene transfer by conjugation of megaplasmids >40 kb (35). Because the CRISPR system identified in this study is encoded within a putative mobile genetic element, we propose that an additional mechanism of mobilization to other methicillin-susceptible Staphylococcus spp. is plausible.

This novel subtype of SCCmecV was found in only 4 of the 6 LA-MRSA isolates identified in this study. One isolate not containing this novel SCCmec subtype (08 BA 08100) could also be distinguished by a different but closely related spa type (t1250) (Table 1) and variant PFGE fingerprint (Figure 2) when compared with the other LA-MRSA isolates, which suggests that at least 2 epidemiologically different strains of LA-MRSA circulate in Saskatchewan. The other LA-MRSA isolate that did not contain this novel SCCmec element was obtained in Ontario. However, this isolate was the same spa type (t034) and was closely related, according to PFGE, to the LA-MRSA isolates identified in Saskatchewan. Therefore, in addition to PFGE and spa typing, SCCmec subtyping could provide a useful epidemiologic tool for surveillance, outbreak investigations, or traceability studies of this emerging strain. For detection of this SCCmecV subtype (tentatively designated V.2.1.2; Vb), we propose using primer set 1 (spanning orfX into Sk02 in the J3 region) and primer set 7 (spanning Sk20 into cas1 in the J1 region) (Table 4).

Visual comparison of PFGE fingerprints from this study with those reported from patients from the Dominican Republic and the United States (northern Manhattan, New York, NY) (36), showed substantial variations in fingerprint patterns, as well as related but different spa types. These variations suggest further molecular and geographic diversity of these LA-MRSA strains on a global scale.

Because cases of LA-MRSA infections have only recently been identified in Canada, additional surveillance efforts are required to monitor the emergence and clinical relevance of this MRSA strain in Canada, including communities, the environment, livestock, farmers, and production facility workers. Whether these strains pose a major threat to human health in light of the low livestock density and continued spread of epidemic hospital and community strains of MRSA in Canada remains unknown.