Setting

The investigation focused on an area in east-central Saskatchewan. This region consisted of a city of ≈7,127 persons that was serviced by a hospital with a central laboratory in which the MRSA strains were identified. This locale also contained a number of community health centers and a long-term care facility housing ≈100 persons. The region also included a number of smaller communities that consisted of small rural First Nations and Metis communities (aboriginal populations) and small towns.

Case Definitions

Surveillance for MRSA was laboratory-based and involved nonrepeat MRSA cases. Case-patients were residents of east-central Saskatchewan with laboratory-confirmed MRSA infections identified from January 1, 1999, to March 31, 2002, by the local hospital or provincial laboratory. All MRSA isolates identified by the hospital laboratory were subsequently confirmed at the provincial laboratory by using standard protocols. Residential status was determined by the location of the patient's treatment facility because case-patient address information was unavailable. General patient demographic information was collected (date of birth, sex, date of sample collection, and invasiveness) for all case-patients. Invasive infections were defined according to the guidelines from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance Program and included obtaining isolates from a normally sterile site, such as blood, cerebrospinal fluid, pleural fluid, peritoneal fluid, pericardial fluid, surgical aspirate, bone, joint fluid, or internal body site (www.cdc.gov/ncidod/dbmd/abcs/meth-case.htm). Antimicrobial susceptibility testing by microbroth dilution was performed according to NCCLS recommendations (13). Breakpoints used for mupirocin were as follows: susceptible, MIC <4 mg/L; low-level resistance, MIC ≥4 and <256 mg/L; high-level resistance, ≥256 mg/L (14).

Molecular Characterization of MRSA Strains

mecA and nuc genes from MRSA isolates were coamplified with a multiplex real-time polymerase chain reaction (PCR) assay. Nucleic acid was isolated from 4–5 colony picks by boiling in a 2% (wt/vol) homogeneous suspension of Chelex 100 resin (Bio-Rad Laboratories Ltd, Mississauga, Ontario, Canada). Primer and probe sequences with their reaction concentrations are shown in Table 1. Master mix was composed of the Applied Biosystems TaqMan PCR Core kit (Applied Biosystems, Foster City, CA, USA) that uses reaction concentrations of: 1× PCR buffer A, 4.0 mmol/L MgCl₂, 200 μmol/L dATP, 200 μmol/L dCTP, 200 μmol/L dGTP, 400 μmol/L dUTP, 1.25 U AmpliTaq Gold, and 0.5 U uracil-DNA-N-glycosylase (UNG) for carryover prevention. Thermal cycling and data collection were performed on an ABI Prism 7700 Sequence Detector using the following conditions: 2 min at 50°C, 10 min at 95°C, followed by 55 cycles of 95°C for 15 s and 60°C for 1 min. Amplification was confirmed for each target by the generation of a sigmoid amplification plot.

lukF-PV and lukS-PV detection was carried out by using PCR with primers and protocols previously described (15). PCR was used to detect the exfoliative toxin genes eta and etb as previously described (16).

Isolates were subtyped by using pulsed-field gel electrophoresis (PFGE) after digestion with SmaI following the Canadian Standardized Protocol as described previously (17). PFGE-generated DNA fingerprints were digitized and analyzed with BioNumerics Ver. 3.5 (Applied Maths, Sint-Martens-Latem, Belgium) by using a position tolerance of 1.0 and an optimization of 1.0. Relatedness was determined following established criteria with major strain clusters (designated by a letter) grouped with banding patterns of <7 band changes (18). Fingerprints were compared to those in the national MRSA fingerprint database, which comprised of >600 unique MRSA fingerprint types. Isolates with specific DNA profiles were grouped into 1 of 6 Canadian epidemic strains of MRSA (CMRSA-1, CMRSA-2, CMRSA-3, etc.) as previously described (19,20). Multilocus sequence typing (MLST) was conducted on a representative isolate from each of the unique PFGE types as previously described (21). Staphylococcal chromosome cassette (SCC) mec typing was conducted as previously described (22).

Epidemiologic Analysis of MRSA

Figure 1

Figure 1. . Epidemiologic curve showing the emergence of methicillin-resistant Staphylococcus aureus (MRSA) in central-eastern Saskatchewan. A) Number of nonrepeat cases over the length of study; solid bars, cases identified in a long-term...

Before 1999, MRSA was rarely isolated in east-central Saskatchewan. It was identified only 2 other times in persons from this region since 1996. However, in April 1999, a resident of a long-term care facility in the region under study tested positive for MRSA. This patient had recently been hospitalized in Saskatoon, Saskatchewan, and tested positive for MRSA shortly after being transferred back to the long-term care facility. Following this case, an additional 183 nonrepeat MRSA were isolated from infected persons from this region between January 1999 and April 2002. The annual rates of MRSA-related infections in the region under study were 3.1/1,000 persons in 1999, 4.8/1,000 persons in 2000, and 14.6/1,000 persons in 2001. In 1 specific community within the study area with a population of approximately 1,400 persons, MRSA rates were 0/1,000 in 1999, 7.1/1,000 in 2000, and 46/1,000 in 2001. Only a single isolate was considered invasive, having causes a blood infection. Approximately 18% (n = 33) of the cases were identified from a single long-term care facility and made up of >50% of cases identified between April 1999 and June 2000 (Figure 1A). After June 2000, a larger cluster of cases emerged, which peaked in October 2001 and were primarily identified from patients presenting at local health clinics or nursing stations from surrounding communities (Figure 1A).

Molecular Characterization of MRSA

Figure 2

Figure 2. . A) Pulsed-field gel electrophoresis (PFGE) fingerprint of USA400 and PFGE pattern B1. B) Dendrogram showing relationship of the unique fingerprints, along with the PFGE type designation (11) and...

All of the 184 strains were PCR positive for the mecA and nuc genes, respectively. DNA fingerprinting of all strains using PFGE resulted in the identification of 5 major fingerprint patterns labeled pattern A to E (Figure 2). Clonal group A (n = 55) comprised 8 subtypes with subtype A1 representing 86% (n = 47, Canadian Diseases Network (CDN) type 417) of the total, with other A subtypes as follows: A2 (n = 1, CDN type 697); A3 (n = 1, CDN type 695); A4 (n = 2, CDN type 691); A5 (n = 1, CDN type 696); A6 (n = 1, CDN type 550); A7 (n = 1, CDN type 726); A8 (n = 1, CDN type 725). Group B was the most predominant molecular subtype with 68% (n = 126) of the strains in this cluster. This group was comprised of 3 PFGE subtypes labeled B1 (n = 123, CDN type 142), B2 (n = 1, CDN type 378), and B3 (n = 2, CDN type 418). Three additional unique PFGE fingerprint types were labeled C1 (n = 1, CDN type 494), D1 (n = 1, CDN type 334), and E1 (n = 1, CDN type 147).

Characterization of Clonal Group A Isolates

Figure 3

Figure 3. . Age distribution of patients with cases caused by each clonal type A (white bars) or type B (black bars).

Clonal group A isolates were first identified on April 12, 1999, from a patient in a long-term care facility who had been transferred from a hospital in Saskatoon, Saskatchewan. A rapid increase in cases was related to this clonal group from persons from the long-term care facility (n = 33, 60%) as well as community health centers (n = 22, 40%) during the next few months (Figure 1B). Cases related to this group peaked June 1999; however, isolates continued to be identified over the course of this study from both patients in long-term care facilities and community health centers (Figure 1). Clonal group A strains were found to cause a large number of noninvasive infections. Only a single isolate was identified as causing an invasive blood infection. A slightly higher number of female patients 58% (n = 32) were identified with clonal group A–related infections. Approximately 64% (n = 35) of these strains were identified from persons >59 years of age, although 29% (n = 16) of cases were identified in persons <10 years of age (Figure 3). Of the 22 cases reported from the community health centers, 73% (n = 16) were <18 years of age. Comparison of the representative PFGE clonal group A1 fingerprint pattern to the Canadian National Fingerprint Database showed that these strains were similar to the previously identified epidemic MRSA-labeled CMRSA-2 (19,20). A representative strain from each of the unique fingerprint patterns from the A clonal group was typed by using MLST, and all were found to be of the sequence type ST5. All of the unique A subtypes as well as a 25% (n = 12) sample of strains that were of the A1 subtype did not harbor the lukF-PV, lukS-PV, eta, and etb toxin genes. SCCmec typing of 4 A1 subtypes and subtypes A2, A3, A4, A5 showed all contained SCCmec IIIb. Most clonal group A isolates were resistant to ciprofloxacin, erythromycin, gentamicin, tetracycline, mupirocin, and fusidic acid (Table 2).

Characterization of Clonal Group B Isolates

The first reported case of infection due to clonal group B occurred 16 months after the description of first reported MRSA infection on August 31, 2000, and the cases continued to increase with a peak of occurring in October 2001 (Figure 1B). Clonal group B correlated with a large number of noninvasive infections reported from community health centers, and only 1 report was made of a long-term care patient with MRSA type 0142 infection. The distribution by sex was similar; 49% of patients were female (n = 62) and 51% were male (n = 64). More than 67% (n = 85) of these strains were identified from persons <20 years of age, although ≈17% (n = 21) of cases were identified in persons >40 years (Figure 3).

The PFGE B1 fingerprint pattern (CDN type 142) was indistinguishable from the USA CA-MRSA strain labeled USA400 (Figure 2) (21). MLST was conducted on 1 representative strain from each clonal group B subtype, and all were the same sequence type (ST1). In addition, these MRSA strains were shown to contain SCCmec type IV. A selection of ≈25% of the PFGE type 142 strains (n = 33), including all of the unique fingerprint patterns B2 and B3, was shown by PCR to harbor the lukF-PV and lukS-PV genes, and none of the strains tested harbored the eta and etb toxin genes. Clonal group B strains were susceptible to most antimicrobial agents tested; however, 40% and 55% of the isolates displayed resistance to erythromycin and high-level resistance to mupirocin, respectively (Table 2).

Characterization of Other Molecular Subtypes

Three other unique PFGE subtype groups were identified; these were composed of single isolates labeled C1, D1, and E1, respectively (Figure 2). None of these isolates carried the lukF-PV and lukS-PV toxin genes or the exfoliative toxins. Similar to the PFGE B clonal group, these strains remained susceptible to most non–β-lactam drugs tested (data not shown).