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Journal of Clinical Microbiology, December 2005, p. 6147-6149, Vol. 43, No. 12
0095-1137/05/$08.00+0 doi:10.1128/JCM.43.12.6147-6149.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Evelyn Nagle,1
Michael R. Mulvey,2
Paul N. Levett,1 and
Greg B. Horsman1
Provincial Laboratory, Saskatchewan Health, Regina, Saskatchewan, Canada,1 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada2
Received 21 September 2005/ Accepted 4 October 2005
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ABSTRACT |
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The discovery of Panton-Valentine leukocidin (PVL), encoded by lukF-PV and lukS-PV genes, was reported in the 1930s (16), and PVL has been associated with 1.6% of S. aureus strains in England and Wales in 2002 (8). Recently, MRSA strains producing PVL have been associated with an increase in severe skin infections and a new syndrome of community-associated necrotizing pneumonia in children (19). The PVL genes are carried on the phage 
SLT (15), but to date, PVL has not been identified with epidemic or endemic hospital-acquired MRSA strains (12).
With the emergence of the USA300 and USA400 CA-MRSA strains in Canada, also known as CMRSA10 and CMRSA7, respectively (13; M. Gilbert, J. Siushansian, J. MacDonald, D. Gregson, S. Elsayed, K. Zhang, K. Laupland, M. Louie, T. Louie, D. Nielsen, G. Keays, A. Honish, D. Gravel, M. Mulvey, J. Gillespie, and J. Conly, Abstr. AMMI Canada—CACMID 2005 Annu. Conf., abstr. G2, 2005), the challenge lies with laboratories to efficiently identify these strains for surveillance, infection control, and treatment protocols to limit the dissemination of CA-MRSA (11, 19, 20). Conventional PCR detection of the lukF-PV and lukS-PV genes of the PVL-encoding operon (17) allowed the identification of PVL-containing strains (11). However, real-time qualitative PCR detection of PVL genes (6, 9, 14) can reduce the turnaround time significantly. In this report, we evaluated a high-throughput real-time PCR detection of PVL-encoding genes, multiplexed for simultaneous amplification of mecA (for detection of methicillin resistance) and nuc (for identification of S. aureus) (4). This triplex TaqMan assay is applied to the analysis of MRSA isolated from clinical specimens as part of a public health surveillance of MRSA.
Clinical MRSA isolates (n = 1,557) were collected throughout Saskatchewan, Canada, primarily from skin and soft tissue infections from both healthcare and community settings between 1999 and 2005. All S. aureus isolates were considered oxacillin resistant by growth on Mueller-Hinton agar (containing oxacillin [6 µg/ml] and 4% [wt/vol] NaCl) and by an automated sensitivity assay (Microscan; Dade Behring Canada, Inc., Mississauga, Ontario, Canada) (5) prior to PCR analysis. Nucleic acids from S. aureus were prepared by heating four to five colony picks from an overnight plate culture in 2% (wt/vol) Chelex 100 resin slurry (Bio-Rad Laboratories, Ltd., Mississauga, Ontario, Canada) for 10 min at 100°C. MRSA was confirmed by the detection of mecA and nuc by TaqMan duplex PCR or triplex PCR as described below. Primer and probe sequences for PVL (Table 1) were designed using Primer Express (ver. 2.0; Applied Biosystems, Mississauga, Ontario, Canada) and Oligo 6 (ver. 6.6.7.0; Molecular Biology Insights, Inc., Cascade, Colo.). A DNA alignment of publicly available lukF-PV and lukS-PV gene sequences from S. aureus and phage SLT (GenBank accession no. AB006796, X72700, AB009866, and AB045978) was considered to select a conserved target that spanned both genes of the operon. Specificity of primers and probe was assessed in silico against the NCBI (GenBank) nucleotide sequence databases by using BLASTN (2). Initially, PVL was detected with a single-target TaqMan PCR assay by using a dual-labeled probe with TET (6-carboxytetrachlorofluorescein) as the reporter and TAMRA (6-carboxytetramethylrhodamine) as the quencher (Applied Biosystems). Single-target PVL reactions were run simultaneously with the duplex assay for mecA and nuc in an ABI Prism 7700 sequence detector (Applied Biosystems) on the same plate, using a TaqMan PCR core reagent kit (Applied Biosystems) at 4.0 mM MgCl2. Thermal cycling was performed under the following conditions: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
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To validate the PVL TaqMan primer and probe sequence design, comparative results using the conventional PCR and the single-target TaqMan assay were collected for 103 clinical MRSA isolates. Results obtained for these isolates by use of the two methods were in agreement (100% sensitivity, 100% specificity), with 54 isolates yielding positive amplification.
During a 15-month period, 752 clinical MRSA isolates were tested by the single-target PVL assay in conjunction with the duplex assay for mecA and nuc, of which 321 (43%) were PVL positive. The performance of the triplex TaqMan assay was subsequently compared directly to that of the single-target PVL assay by using the results obtained from 98 different isolates. The results of the two TaqMan assays showed 100% concordance. Reference strains not containing PVL (MRSA [ATCC 43300], S. aureus [ATCC 29213], and methicillin-sensitive S. epidermidis [ATCC 12228]) did not show amplification with the PVL primers and probe. Reproducibility of the triplex PVL assay was confirmed with the consistent amplification of a clinical isolate of S. aureus (BA37594) on 31 separate occasions. Subsequently, 516 consecutive clinical isolates have been assayed with the triplex PCR. PVL was detected in 259 (50%) of these isolates.
CA-MRSA is rapidly becoming a public health concern in many parts of the world, including Canada (7, 13, 21). In Saskatchewan, CA-MRSA, described as similar to USA400 by pulsed-field gel electrophoresis and harboring staphylococcal chromosome cassette mec (SCCmec) type IV and PVL, has been identified in certain communities (13), and this strain is now widespread in the province (R. R. McDonald, K. Keller, L. Snook, E. Nagle, C. Selin, M. Mulvey, N. Antonishyn, P. N. Levett, and G. Horsman, abstr. 72nd CACMID Conjoint Meet. Infect. Dis., abstr. LB7, 2004). Most infections with PVL-producing CA-MRSA involve skin lesions, but there is also a proportion that present as rapidly progressive necrotizing pneumonia (22, 23). Thus, PVL is thought to be an important virulence factor in these infections as well as a stable marker for CA-MRSA (19). For these reasons, it is important for clinicians to be familiar with the incidence of CA-MRSA in their communities, as prevalence can vary from region to region. Many CA-MRSA soft tissue infections can be treated with incision and drainage, without antibiotic treatment (10). Moreover, since CA-MRSA has a unique antibiotic resistance profile (13, 21), knowledge of prevalence can inform empirical antibiotic therapy in more-severe cases that are handled as outpatients (3). This necessitates surveillance for PVL-containing MRSA by use of the most efficient laboratory methods available.
Real-time PCR allows for rapid detection of target sequences with high throughput and is the best-suited technology for this and other applications. A design of a FAM/VIC/TET triplex PCR on an Applied Biosystems 7700 instrument was contemplated; however, because the potential for cross talk due to emission spectrum overlap for these three dyes was high, the protocol was abandoned. Indeed, following amplification we observed the detection of fluorescence in the TET (PVL) layer of PVL-negative MRSA isolates. Furthermore, fluorescence was detected in the TET layer of MRSA reactions containing only FAM- and VIC-labeled probes (data not shown). Contrary to another report (14), the reproducibility of these observations did not allow us to pursue a FAM/VIC/TET triplex PCR application. Use of Cy5 as a reporter dye with the Applied Biosystems 7500 instrument was an alternative approach. The change has allowed the assay for mecA, nuc, and PVL to be less laborious, using only one master mix that also generates savings in costs associated with the assay.
PVL-containing MRSA is an emerging pathogen in the province of Saskatchewan and elsewhere in Canada. The multiplex reaction described in this report should allow rapid detection of CA-MRSA strains by using the presence of the PVL toxin as a marker. In addition, this assay could be used to assess the prevalence of the PVL toxin genes in the general S. aureus population.
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ACKNOWLEDGMENTS |
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We acknowledge the technical assistance of the Provincial Laboratory Bacteriology staff and of Carrie Selin.
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FOOTNOTES |
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Present address: Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada.
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REFERENCES |
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SLT. Gene 268:195-206.[CrossRef][Medline]
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