Novel Multiplex PCR Assay for Simultaneous Identification of Community-Associated Methicillin-Resistant Staphylococcus aureus Strains USA300 and USA400 and Detection of mecA and Panton-Valentine Leukocidin Genes, with Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

Sequence alignment and primer design.

Primers for 16S rRNA (Stapy756-F and Staph750-R) (15), thermostable nuclease (nuc) (Nuc-1 and Nuc-2) (15), mecA (mecA147-F and mecA147-R) (14), and PVL genes lukS-PV/lukF-PV (Luk-PV-1 and Luk-PV-2) (7) were as previously described. New sets of primers for strains USA300 and USA400 and for prophage φSa2usa/φSa2mw were designed based on comprehensive analysis and alignment of individual Staphylococcus sp. genomes currently available in the GenBank database. Gene targets for each primer pair are as follows: USA300 strain primers arcA-F (5′-GCAGCAGAATCTATTACTGAGCC-3′) and arcA-R (5′-TGCTAACTTTTCTATTGCTTGAGC-3′) target the arcA gene on the arginine catabolic mobile element (ACME); USA400 strain primers MW756-F (5′-TGGTTAGCTATGAATGTAGTTGC-3′) and MW756-R (5′-GTCCATCCTCTGTAAATTTTGC-3′) target the gene locus MW0756 on νSa3 of strain MW2; and φSa2mw/φSa2usa prophage primers phi-int-F4 (5′-CAAATTTTGAAAACTTTACGC-3′) and phi-int-R4 (5′-TCCAGGATTAAAAGAAGCG-3′) target the MW1409 gene locus of the USA400 MW2 strain.

Development of an M-PCR assay for typing MRSA isolates and distinguishing USA300 and USA400 strains.

The assay specifically involved targeting the Staphylococcus genus-specific 16S rRNA gene sequence (serving to distinguish Staphylococcus from other bacteria and acting as an internal PCR control), the S. aureus-specific nuc gene, the methicillin resistance determinant mecA, the PVL genes, the phage marker MW1409, the USA400 genomic island gene locus (MW0756), and the USA300 ACME cassette gene (arcA). Amplification in a single M-PCR produced distinct bands corresponding to molecular sizes of 147, 220, 279, 372, 433, 513, and 756 bp for mecA, MW1409, nuc, MW0756, PVL, arcA, and 16S rRNA, respectively (Fig. 1).

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FIG. 1.

Novel multiplex PCR assay identifying USA300 and USA400 community-associated MRSA strains, detecting PVL and mecA genes, and simultaneously discriminating S. aureus from CoNS. The optimized M-PCR was performed as follows: 4.65 μl of template DNA was prepared as previously described (15) in a 25-μl final reaction volume containing 0.30, 0.3, 0.4, 0.32, 0.16, 1.17, and 0.16 μM for each of the 16S rRNA, arcA, lukS-PV/lukF-PV, MW0756, nuc, MW1409, and mecA primers, respectively, with the thermocycling conditions set at 94°C for 4 min followed by 10 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 45 s and another 25 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 45 s. The PCR amplicons were visualized using a UV light box after electrophoresis on a 2% agarose gel containing 0.5 μg/ml ethidium bromide. Lane 1, strain ATCC 29213 (PVL⁻ MSSA); lane 2, strain ATCC 49775 (PVL⁺ MSSA); lane 3, Canadian epidemic MRSA control strain CMRSA2 (PVL⁻ non-USA300 and non-USA400 MRSA); lane 4, strain C1538 (PVL⁺ non-USA300 and non-USA400 MRSA); lane 5, strain C2901 (PVL⁻ USA400); lane 6, Canadian epidemic CA-MRSA USA400 control strain CMRSA7 (PVL⁺ USA400); lane 7, Canadian epidemic CA-MRSA USA300 control strain CMRSA10 (PVL⁺ USA300); lane 8, strain CNS99-PF5 (PVL⁻ and arcA⁻ MS-CoNS); lane 9, strain CNS99-PF7 (PVL⁻ but arcA⁺ MS-CoNS); lane 10, strain CNS99-PF6 (PVL⁻ and arcA⁻ MR-CoNS); lane 11, strain CNS99-PF8 (PVL⁻ but arcA⁺ MR-CoNS); lanes M, 1 kb Plus DNA ladder (Invitrogen). Refer to Table 1 for details of each strain.

Validation of M-PCR assay.

The new multiplex PCR assay was validated using 42 representative MRSA/MSSA control strains and 6 ACME-positive (ACME⁺) or ACME-negative (ACME⁻) CoNS control strains that had undergone detailed phenotypic and molecular characterization, including analysis of carriage of PVL (7) and other genes, pulsed-field gel electrophoresis (PFGE) fingerprinting (9), SCCmec typing (14), spa typing (5, 10), and MLST (2) and eBURST (3, 11) analyses (Table 1). The MRSA/MSSA strains differed in their genotypic characteristics and represented 10 major clonal complex groups found in the worldwide MLST collection. The assay was capable of accurately and reproducibly discriminating USA300 from USA400 and other MRSA and MSSA strains while simultaneously detecting PVL genes and φSa2mw/φSa2usa phages, with a resultant 100% concordance to genotypic features in all these control strains (Table 1). There were seven PVL⁺ MRSA and nine PVL⁺ MSSA strains, belonging to non-USA300/non-USA400 strains with well-diversified genomic backgrounds, and all were positive for the PVL genes but negative for the φSa2mw/φSa2usa phage marker, suggesting that PVL genes in these strains may be carried by phages/plasmids other than φSa2mw/φSa2usa. More interestingly, there were two strains (with non-USA300/non-USA400 PFGE profiles), one a PVL⁻ MRSA strain (CMRSA5) and one a PVL⁺ MSSA strain (SAF516), that were positive for the phage marker. Since this primer pair is a marker for the PVL-bearing phage φSa2mw/φSa2usa in USA400/USA300, this result suggests that variations of these particular phages can also be present in S. aureus strains other than USA300 and USA400 with or without the PVL genes. Further studies are required to better understand this observation.

Applicability and accuracy of M-PCR.

To address the applicability and accuracy of the M-PCR assay, we further applied our M-PCR assay to test a total of 1,133 local clinical MRSA isolates randomly selected from our Calgary frozen clinical isolate stock collection for the 18-year period from 1989 to 2006. We were able to accurately identify and classify all strains with available PFGE data, including 54 PVL⁺ USA300, 17 PVL⁺ USA400, 35 PVL⁻ USA400, 40 PVL⁻ CMRSA2, and 34 PVL⁻ non-USA300/non-USA400/non-CMRSA2 MRSA strains. We were also able to clearly classify the remaining randomly chosen strains, including 514 S. aureus and 439 CoNS isolates. Once again, we noted that 10 (1.9%) of the isolates, including 5 PVL⁻ MRSA and 5 PVL⁻ MSSA isolates, were positive for the phage gene and yet did not belong to either USA300 or USA400. There was also one MRSA isolate that was positive for the PVL genes but negative for the φSa2mw/φSa2usa gene. Among 439 CoNS isolates tested, there was a 100% concordance with phenotypic susceptibility to methicillin, with 214 of the isolates being methicillin-susceptible CoNS (MS-CoNS) and 225 being methicillin-resistant CoNS (MR-CoNS). Of the MS-CoNS isolates, 75 (35.0%) of them were positive for arcA, while 93 (41.3%) of the MR-CoNS isolates were arcA positive. None of the CoNS isolates tested carried PVL, φSa2mw/usa phage, or USA400 marker MW0756 gene loci.

Our assay is capable not only of (i) accurately identifying and differentiating USA300 and USA400 strains but also of simultaneously detecting (ii) mecA to discriminate MRSA from MSSA and (iii) 16S rRNA and nuc to discriminate S. aureus from CoNS and of detecting (iv) PVL genes and a φSa2mw/usa-specific gene to determine whether the isolates/strains carry PVL genes and whether the PVL genes are carried by the φSa2mw/φSa2usa phage or other prophages in the population. Our M-PCR assay may also facilitate monitoring of the dynamic exchanges or evolution of genes among strains of MRSA, MSSA, and CoNS. This assay is based on the concept of certain strains carrying unique/specific genes/phages. However, since movement of these genes, phages, and other genetic elements is dynamic, the definition of a strain, as determined by the use of our assay for detection of gene targets uniquely present in individual CA-MRSA strains, may require reconsideration over time.