New Real-Time PCR Assay for Rapid Detection of Methicillin- Resistant Staphylococcus aureus Directly from Specimens Containing a Mixture of Staphylococci

The reference strains used for this study are listed in Tables 1 and 2. The staphylococcal clinical isolates used for this study are part of the SENTRY program collection and several supplier's collections. The S. aureus clinical isolates originated from Africa (n = 15) as well as the following 29 countries: Albania (n = 2), Argentina (n = 50), Australia (n = 71), Austria (n = 2), Belgium (n = 3), Brazil (n = 78), Canada (n = 601), Chile (n = 42), China (n = 70), Denmark (n = 33), Egypt (n = 1), France (n = 36), Germany (n = 27), Greece (n = 4), Ireland (n = 5), Israel (n = 19), Italy (n = 61), Japan (n = 62), Mexico (n = 1), The Netherlands (n = 179), Poland (n = 33), Portugal (n = 76), Singapore (n = 20), Slovenia (n = 1), Spain (n = 30), Switzerland (n = 13), Turkey (n = 28), United Kingdom (n = 10), and United States (n = 535). The CoNS clinical isolates originated from the following eight countries: Argentina (n = 13), Canada (n = 95), China (n = 7), Denmark (n = 21), France (n = 4), Japan (n = 28), United Kingdom (n = 44), and United States (n = 37). Confirmation of the identification of the staphylococcal strains was done by use of the MicroScan WalkAway Panel Type Positive Breakpoint Combo 13 system when required (Dade Behring Canada Inc., Mississauga, Ontario, Canada).

The MICs for oxacillin were either provided by the strain suppliers or determined in our laboratory by the Etest method (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions. The results were interpreted according to the standards of the NCCLS (37). S. aureus strains ATCC 29213 and ATCC 43300 were used as quality controls for antimicrobial susceptibility testing.

The primers used to amplify and sequence the SRE junctions (MREJs) of various MRSA strains are listed in Table 3. The MREJ comprises the right extremity of SCCmec, the SCCmec integration site, and the orfX gene.

The MREJ fragments to be sequenced were amplified on a PTC-200 thermocycler (MJ Research Inc., Watertown, Mass.), using purified genomic DNA. Genomic DNAs were purified by use of a Gnome kit (Qbiogene Inc., Carlsbad, Calif.) according to the manufacturer's instructions. Depending on the MRSA strain, amplification products of 1.2 or >8 kb were obtained. Amplification products of 1.2 kb were amplified as previously described (29), whereas those of >8 kb were amplified with Herculase Enhanced DNA polymerase (Stratagene, La Jolla, Calif.). Purification of the amplification products and sequencing reactions were performed as previously described (29).

A multiple nucleotide sequence alignment of the MREJ sequences of several staphylococcal species obtained for this study or available from public databases was performed with the Pileup program from the GCG package (version 10; Accelrys, San Diego, Calif.). This alignment allowed the design of a set of PCR primers specific to the various SRE sequences (mecii574, meciii519, meciv511, mecv492, and mecvii512) as well as a primer specific to S. aureus orfX (Xsau325). This set of primers was used in combination with three MBPs (XsauB5-FAM, XsauB8-FAM, and XsauB9-FAM) targeting orfX sequences within the MREJ amplification products, allowing the detection of all S. aureus orfX variants. A fourth MBP (PSARM-TET) was used to detect pSARM internal control amplification products. MBPs were designed as previously described (4). Primers were designed with the help of Oligo Primer Analysis software, version 6.22 (Molecular Biology Insights, Cascade, Colo.). Primers were synthesized with a DNA synthesizer (model 391; PE Applied Biosystems, Foster City, Calif.) and MBPs were obtained from Biosearch Technologies (Novato, Calif.). The PCR primers and probes used for this study are listed in Table 3 and are shown in Fig. 1.

An internal control was constructed as described previously (28). A 324-bp DNA fragment consisting of a 276-bp sequence not found in MRSA flanked by the sequences of the meciv511 and mecv492 primers (Table 3) was used as a template for the internal control. This fragment was cloned into the pCR2.1 vector (Invitrogen, Burlington, Ontario, Canada) and isolated as previously described (28). The purified recombinant plasmid, named pSARM, was linearized with BamHI (New England Biolabs, Mississauga, Ontario, Canada) and serially diluted. The concentration of the linearized plasmid was optimized to permit the amplification of the 324-bp internal control product without a significant detrimental effect on MRSA-specific amplification.

For all bacterial strains, amplification was performed with either purified genomic DNAs or crude DNA extracts prepared from bacterial suspensions whose turbidity was adjusted to that of a 0.5 McFarland standard, which corresponds to approximately 1.5 × 10⁸ bacteria per ml. Crude DNA extracts were prepared for PCRs by a rapid DNA extraction method (Infectio Diagnostic Inc., Ste-Foy, Canada) (28). One microliter of purified genomic DNA or of a crude DNA extract was transferred directly to a 24-μl PCR mixture containing oligonucleotide primers and probes (Table 3), four deoxyribonucleoside triphosphates (Pharmacia Biotech, Baie d'Urfé, Québec, Canada), a 10 mM Tris-HCl buffer (pH 9.0), 4.75 mM MgCl₂, 2.5 mg of bovine serum albumin per ml, and 1.25 U of Taq DNA polymerase (Roche, Mississauga, Ontario, Canada) combined with a TaqStart antibody (Clontech Laboratories, Palo Alto, Calif.). The internal control (pSARM) was included in the PCR assay to ensure that there was no significant PCR inhibition by the test sample. The thermal cycling protocol was as follows: 3 min at 95°C for initial denaturation followed by 48 cycles of three steps consisting of 5 s at 95°C for denaturation, 15 s at 60°C for annealing, and 20 s at 72°C for extension. Real-time detection of the PCR products was performed on a Smart Cycler (Cepheid, Sunnyvale, Calif.) by measuring the fluorescence signal emitted by the MBPs hybridized to their targets at the end of each annealing step. The specificity and ubiquity (i.e., the ability to detect all or most MRSA strains) (5) of the PCR assay were verified by use of a panel of 22 gram-negative bacterial species, 40 gram-positive nonstaphylococcal bacterial species, 14 species (212 strains) of MRCoNS, 27 species (74 strains) of MSCoNS, 569 MSSA isolates, and 1,657 MRSA strains from various geographic areas. For determination of the analytical sensitivity of the PCR assay, 1-μl samples of serial twofold dilutions of purified genomic DNA (ranging from 64 to 1 genome copy per μl) from MRSA strains of different MREJ types were used to determine the minimal number of genomes which were detected. To verify that MRSA amplification was not inhibited in the presence of DNAs from MSSA, MRCoNS, and MSCoNS, we performed the following experiment. The equivalent of 10 genome copies of DNA from MRSA strain ATCC 43300 (carrying MREJ type ii) per PCR was amplified by the real-time PCR assay in the presence of 0, 10, 10², 10³, and 10⁴ genome copies of DNA (per PCR) from either (i) MSSA strain ATCC 29213 (MREJ negative), (ii) methicillin-resistant Staphylococcus epidermidis strain ATCC 35983 (carrying MREJ type ii), or (iii) methicillin-susceptible S. epidermidis strain ATCC 14990 (MREJ negative).

The MREJ types of the MRSA strains described in this study were determined by sequence analysis or by examining the PCR amplification products generated by the multiplex PCR assay described above by standard agarose gel electrophoresis (44). The SCCmec types and subtypes of the MRSA strains described in this study were identified by using a previously described typing method (38). Primers mecIVc70 (5′-TGGGGTATTTTTATCTTCAACTC-3′) and mecIVc1079 (5′-TGGGATTTTAAAGCAGAATATCA-3′) were designed to identify SCCmec type IVc based on the SCCmec sequence of MRSA strain MR108 (25). Primers mecIVd26 (5′-ACGGGAGATTAGGAGATGTTAT-3′) and mecIVd307 (5′-CAGCCATCAATTTTGTTTCACC-3′) were designed to identify SCCmec type IVd based on the SCCmec sequence of MRSA strain JCSC 4469 (GenBank accession number AB097677 ).

The minimal number of CFU that can be detected in nasal specimens was evaluated by using 18 nasal swabs obtained from nine volunteers (2 nasal swabs/volunteer) who were not colonized by MRSA. Nasal specimens were collected with a collection and transport system for aerobes (Venturi Transystem; Copan Canada, Richmond Hill, Ontario, Canada). A swab was carefully inserted a short distance into each nostril and gently rotated for 5 s. Swabs were inserted into the transport medium immediately after samples were obtained. Each swab was streaked directly on mannitol salt agar to determine the presence of MSSA, MRSA, MRCoNS, or MSCoNS. The agar medium was examined after 1 and 2 days of incubation for typical staphylococcal colonies. The identification of suspicious staphylococcal colonies was based on catalase production, slide agglutination (Staphaurex; Murex Biotech Limited), and the tube coagulase test. Identification of and oxacillin MIC determinations for the staphylococcal isolates were performed as described above. None of the specimens contained MRSA isolates, whereas 10 specimens contained MSCoNS isolates, 4 specimens contained MRCoNS isolates, and 4 specimens contained both MSSA and MSCoNS isolates. Nasal specimens from the 18 swabs were resuspended in buffer and pooled. The pooled MRSA-negative specimens, which contained a mixture of MSSA, MRCoNS, and MSCoNS, were divided into 18 aliquots. The samples were spiked with serial 10-fold dilutions of MRSA strain ATCC 43300 during the logarithmic phase of growth (optical density at 600 nm of ∼0.6) in phosphate-buffered saline. Each 10-fold dilution was added directly to the clinical specimens and processed prior to PCR amplification by a rapid DNA extraction method (28). The number of CFU was estimated by standard plating procedures.

GenBank accession numbers for the S. aureus MREJ sequences are as follows: for strain ATCC 33592, AY267373 ; for strain ATCC BAA-40, AY267374 ; for strain CMRSA-1, AY267375 ; for strain CCRI-1263, AY267376 ; for strain CCRI-1311, AY267377 ; for strain CCRI-1331, AY267379 ; for strain CCRI-1377, AY267380 ; for strain CCRI-2025, AY267381 ; for strain CCRI-8895, AY267382 ; for strain CCRI-8903, AY267383 ; and for strain CCRI-9583, AY267384 .

A molecular method named mec right extremity polymorphism (MREP) typing, which takes advantage of the polymorphism among SRE sequences, was previously developed (17, 23). This method combines primers specific to each distinct SRE sequence with a primer specific to the S. aureus chromosome located to the right of the SCCmec integration site to detect the different SCCmec types encountered. MREP types i, ii, and iii were defined according to the respective SCCmec types I, II, and III (17, 23). By using this strategy, we have developed a new set of primers for the specific identification of MRSA by using the known SRE sequences of SCCmec types I, II, III, IVa, IVb, and IVc (2, 23-25, 32, 34). The first primer, mecii574, recognizes the right extremity sequences of SCCmec types I, II, IVa, IVb, and IVc. The right extremities of SCCmec types II, IVa, IVb, and IVc are identical but differ from the right extremity of SCCmec type I by a 102-bp insertion (2, 23-25, 32, 34). The second primer, meciii519, was specific to the right extremity sequence of SCCmec type III. These two primers were used in combination with a primer specific to the S. aureus orfX gene, located to the right of the SCCmec integration site (Xsau325). We used this set of primers to amplify the MREJs of a variety of MRSA strains. We found that 15 of 206 MRSA strains tested could not be amplified with this assay (data not shown). The MREJs of 11 of these 15 strains were sequenced in order to establish the new sequences at the right extremity of SCCmec to allow the development of a PCR assay to detect more MRSA strains. The MREJ sequences obtained for these 11 MRSA strains were compared to sequences available from public databases.

Portions of the MREJ sequence corresponding to the orfX gene for these 11 MRSA strains had identities ranging from 97 to 100% with the publicly available orfX sequences of S. aureus and from 79.1 to 82.5% with those of other staphylococcal species (S. epidermidis, S. hominis, and S. haemolyticus). The characteristic right chromosome-SCCmec junction attL sequence, which comprises the inverted repeat IRscc-R and the direct repeat DRscc (23), was present within the 3′ end of the orfX gene for all 11 strains. However, the DNA sequence within the right extremity of SCCmec was shown to be very different from those of types I, II, III, IVa, IVb, and IVc which were previously described (2, 23-25, 32, 34). Three novel SRE sequences were characterized. These sequences were designated MREJ types iv, v, and vii. The MREJs comprising distinct MREP types were named according to the previously described MREP numbering scheme (17, 23). Hence, MREP type i comprises MREJ type i, MREP type ii comprises MREJ type ii, and so on, up to MREP type vii.

The SRE sequences obtained for strains ATCC 33592, ATCC BAA-40, CCRI-1331, CCRI-8895, and CCRI-8903 were nearly identical to each other and exhibited 100% identity with a DNA fragment found within SCCmec type III of MRSA strain 85/2082, which includes the sequence located between the second and third IS431 copies, the inverted repeat sequence of IS431, and part of the IS431 transposase gene sequence (23). However, our sequence data revealed the location of this fragment at the right extremity of SCCmec to be adjacent to the DRscc. These new sequences were designated MREJ type iv because the SRE sequences for these five MRSA strains were different from those of SCCmec type I from MRSA strain NCTC 10442, SCCmec type II from MRSA strain N315, SCCmec type III from MRSA strain 85/2082, SCCmec type IVa from MRSA strain CA05, SCCmec type IVb from MRSA strain 8/6P, and SCCmec type IVc from MRSA strain MR108 (23, 25, 34).

The SRE sequences obtained for strains CCRI-1263, CCRI-1311, CCRI-1377, and CCRI-2025 were nearly identical to each other, were different from those of all four SCCmec types and MREJ type iv, and consequently, were designated MREJ type v. The SRE sequence of MREJ type v did not show any significant homology with any published sequences when compared with publicly available sequences by use of BLAST.

The SRE sequences obtained for strains CCRI-9583 and CMRSA-1 were also different from those of all four SCCmec types and MREJ types iv and v, and consequently, were designated MREJ type vii. Upon a BLAST search, the SRE sequence of MREJ type vii was also shown to be unique, exhibiting no significant homology to any published sequences.

Four of the 15 MRSA strains that were not detected by the PCR assay were not sequenced, as they were shown to carry either MREJ type v or vii based on agarose gel electrophoresis analysis of the amplification products.

The MREJ types defined for several MRSA strains in this study were compared with the SCCmec types established for these strains by using a previously described SCCmec typing method (38). As shown in Table 4, there was no correlation between the MREJ and SCCmec types. The MRSA strain with MREJ type i had SCCmec type I and those with MREJ type iii all had SCCmec type III. It was not possible to identify the SCCmec type of the epidemic strain ATCC BAA-42, which carries MREJ type ii. This strain was shown to carry a class B mec gene complex. However, primers targeting the ccrA and ccrB genes used for this typing method (38) did not generate any amplification products. All other MRSA strains with MREJ type ii carried SCCmec type I, II, or IVd. The two MRSA strains with the new MREJ type vii defined in this study carried SCCmec type II, whereas MRSA strains with the new MREJ type v had either SCCmec type IVa or IVc. MRSA strains with the new MREJ type iv carried SCCmec type III or IVa.

By using the three new SRE sequences for MRSA, we developed a real-time multiplex PCR assay containing six primers and four probes. The detection limit of the assay was determined by using genomic DNAs purified from 24 MRSA strains of different MREJ types and was found to be two to eight genome copies per PCR (Fig. 2). The specificity of the PCR assay was tested by using 40 nonstaphylococcal gram-positive bacterial species, 22 gram-negative bacterial species, 212 MRCoNS isolates, 74 MSCoNS isolates, and 569 MSSA strains from various geographic areas (Tables 1, 2, and 5). None of the nonstaphylococcal MRCoNS and MSCoNS strains tested cross-reacted with the PCR assay. Among the 569 MSSA strains tested, 26 (4.6%) were misidentified as MRSA based on the PCR assay. The ubiquity of the PCR assay was tested with a variety of MRSA strains originating from various geographic areas, including well-known epidemic clones (Tables 1 and 5). Of the 1,657 MRSA strains tested, 1,636 (98.7%) were specifically detected by the PCR assay. Only 1.3% of these MRSA strains, representing a broad variety of origins, were not detected by the assay. An agarose gel analysis of the PCR amplification products revealed that there was no amplification of the genomic DNAs from these 21 strains (data not shown). The amplification of MRSA (∼10 genome copies per PCR) was not inhibited in the presence of increasing concentrations (up to 10⁴ genome copies per PCR) of MSSA, MRCoNS, or MSCoNS (data not shown).

The detection limit of the PCR assay with nasal specimens was evaluated by using 18 MRSA-negative nasal specimens obtained from volunteers. The MRSA-negative specimens, which contained a mixture of MSSA, MRCoNS, and MRCoNS, were spiked with serial 10-fold dilutions of MRSA in the logarithmic phase of growth. The detection limit of the PCR assay was ∼25 CFU of MRSA per nasal swab. There was no PCR inhibition for the 18 nasal samples tested based on the amplification efficiency of the internal control (data not shown).