Novel Multiplex PCR Assay for Detection of the Staphylococcal Virulence Marker Panton-Valentine Leukocidin Genes and Simultaneous

Discrimination of Methicillin-Susceptible from -Resistant Staphylococci

Jo-Ann McClure, John M. Conly, Vicky Lau, Sameer Elsayed, Thomas Louie, Wendy Hutchins and Kunyan Zhang J. Clin. Microbiol. 2006, 44(3):1141. DOI: 10.1128/JCM.44.3.1141-1144.2006.

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JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2006, p. 11411144 0095-1137/06/$08.00 0 doi:10.1128/JCM.44.3.11411144.2006 Copyright 2006, American Society for Microbiology. All Rights Reserved.

Vol. 44, No. 3

Novel Multiplex PCR Assay for Detection of the Staphylococcal Virulence Marker Panton-Valentine Leukocidin Genes and Simultaneous Discrimination of Methicillin-Susceptible from -Resistant Staphylococci
Jo-Ann McClure,1 John M. Conly,1,2,3,4,5 Vicky Lau,1 Sameer Elsayed,2,3,5 Thomas Louie,3,4 Wendy Hutchins,3 and Kunyan Zhang1,2,3,4,5*
Centre for Antimicrobial Resistance, Calgary Health Region/Calgary Laboratory Services/University of Calgary, Calgary, Alberta, Canada1; Departments of Pathology & Laboratory Medicine,2 Microbiology and Infectious Diseases,3 and Medicine,4 University of Calgary, Calgary, Alberta, Canada; and Calgary Laboratory Services, Calgary, Alberta, Canada5

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Received 3 October 2005/Returned for modication 28 November 2005/Accepted 4 January 2006

We developed a new multiplex PCR assay for detection of Panton-Valentine leukocidin virulence genes and simultaneous discrimination of methicillin-susceptible from -resistant staphylococci. This assay is simple, rapid, and accurate and offers the potential for prompt detection of newly emerging community-associated methicillin-resistant Staphylococcus aureus. Historically, methicillin-resistant Staphylococcus aureus (MRSA) has been associated with nosocomial infections (hospital-acquired MRSA [HA-MRSA] strains). However, these organisms have recently emerged as an important cause of community-associated staphylococcal infections (1, 2, 25, 28, 29). Although diversity and variation in their genomic and antibiogram backgrounds exist, virtually all of these newly emerging community-associated MRSA (CA-MRSA) strains carry the Panton-Valentine leukocidin (PVL) virulence genes and possess a novel small mobile staphylococcal cassette chromosome mec (SCCmec) type IV or V genetic element which harbors the methicillin resistance (mecA) gene and which is more easily transferred to other strains of S. aureus than the larger SCCmec types (types I to III) that are prevalent in HA-MRSA strains (25, 28, 30). Panton-Valentine leukocidin is a bicomponent leukocidin encoded by two cotranscribed genes, namely, lukS-PV and lukF-PV (lukS/F-PV), which reside on a prophage and which cause leukocyte destruction and tissue necrosis (6, 22, 27). Until recently, genes coding for PVL were infrequently encountered, being noted in 5% of S. aureus isolates worldwide (10, 12, 15, 28). However, they are found in a very high proportion of newly emerging CAMRSA strains, with rates of 77% to 100%, as reported in various studies (19, 20, 26). The presence of PVL in S. aureus appears to be associated with increased disease severity, ranging from cutaneous infection requiring surgical drainage to severe chronic osteomyelitis and deadly necrotizing pneumonia (7, 10, 11, 14, 17, 18). In the future, screening for the PVL virulence factor in S. aureus may become a routine laboratory procedure (5). Previously described PCR methods for the detection of PVL genes and the methicillin resistance (mecA) gene have required the use of separate assays (15, 24) or involved real-time PCR (4, 13, 16, 21, 23). We recently developed a new conventional multiplex PCR assay for the simultaneous detection of PVL and methicillin resistance (mecA) genes, which could allow the discrimination of MRSA from methicillin-susceptible S. aureus (MSSA) strains if isolates are phenotypically identied as S. aureus prior to PCR testing. The new multiplex PCR assay targets the Staphylococcus genus-specic 16S rRNA gene (which serves as an internal control) with primers Staph756F (5 -AACTCTGTTATTAGG GAAGAACA-3 ) and Staph750R (5 -CCACCTTCCTCCGG TTTGTCACC-3 ) (31), the lukS/F-PV genes (which encode the PVL S/F bicomponent proteins) with primers Luk-PV-1 (5 -ATCATTAGGTAAAATGTCTGGACATGATCCA-3 ) and Luk-PV-2 (5 -GCATCAAGTGTATTGGATAGCAAAA GC-3 ) (15), and the mecA gene (a determinant of methicillin resistance) with primers MecA1(5 -GTAGAAATGACTGAA CGTCCGATAA-3 ) and MecA2 (5 -CCAATTCCACATTGT TTCGGTCTAA-3 ) (31). To ensure that the individual primer pairs were adequate for the amplication of all three gene fragments, a single-target PCR protocol with each individual primer pair was conducted, prior to the multiplex PCR optimization, with 13 control strains (Table 1). Each individual PCR yielded fragments of the expected sizes, i.e., 756, 433, and 310 bp for the 16S rRNA, lukS/F-PV, and mecA genes, respectively (Fig. 1A). The optimized multiplex PCR conditions were obtained by assaying different primer concentrations and other PCR components, as follows: 2 l of template DNA prepared by a previously described boiling method (31) in a 25- l nal reaction volume containing 0.07, 0.08, and 0.24 M for the primers specic for the 16S rRNA, lukS/F-PV, and mecA genes, respectively, with the thermocycling conditions set at 94C for 10 min, followed by 10 cycles of 94C for 45 s, 55C for 45 s, and 72C for 75 s and 25 cycles of 94C for 45 s, 50C for 45 s, and 72C for 75 s. Amplication in a single multiplex PCR produced distinct bands, corresponding to their respective molecular sizes, that were easily recognizable in agarose gels stained with ethidium bromide (Fig. 1B).
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* Corresponding author. Mailing address: Department of Pathology & Laboratory Medicine, University of Calgary, #9-3535 Research Road N.W., Calgary, Alberta T2L 2K8, Canada. Phone: (403) 7703583. Fax: (403) 770-3347. E-mail: kunyan.zhang@cls.ab.ca.

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NOTES TABLE 1. Phenotypic and genotypic characteristics of control strains

J. CLIN. MICROBIOL.

Identication no.

Strain

Strain characteristics a

Phenotypeb (OXA susceptibility)

Genotypec 16S rRNA lukS/F-PV mecA

1 2 3 4 5 6 7 8 9 10 11 12 13
a b

ATCC 51811 ATCC 29213 ATCC 49775 ATCC 25923 CLS-1807 ATCC 33591 HIP 5827 (GISA) JCSC 4469 N02-590 CA05 8/6-3P MR108 ATCC 14990

PVL PVL PVL PVL PVL PVL PVL PVL PVL PVL PVL PVL PVL

( ( ( ( ( ( ( ( ( ( ( ( (

) ) ) ) ) ) ) ) ) ) ) ) )

MSSA MSSA MSSA MSSA MSSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MS-S. epidermidis

S S S S S R R R R R R R S

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PVL ( ), PVL negative; PVL ( ), PVL positive. Oxacillin (OXA) susceptibility was determined with the VITEK system (bioMerieux) and by oxacillin susceptibility screening (4% NaCl Mueller-Hinton agar containing 6 g/ml oxacillin); S, susceptible (MICs, 2 g/ml for S. aureus and 0.25 g/ml for coagulase-negative staphylococci, with no growth on oxacillin screening agar); R, resistant (MICs, 4 g/ml for S. aureus and 0.5 g/ml for coagulase-negative staphylococci, with growth on oxacillin screening agar). c 16S rRNA, Staphylococcus genus specic; lukS/F-PV genes encode Panton-Valentin leukocidin S/F proteins; mecA, determinant of methicillin resistance; , positive for gene; , negative for gene.

To determine assay sensitivity, limiting dilution experiments were performed as described previously (31) with ve representative control strains, including one PVL-negative MSSA strain (ATCC 51811), one PVL-positive MSSA strain (ATCC

49775), one PVL-negative MRSA strain (N315), and two PVLpositive MRSA strains (N02-590 and CA05). This assay was capable of detecting, with reproducibility, a band in ethidium bromide-stained gels at dilutions corresponding to 6 104

FIG. 1. New multiplex PCR assay for simultaneous detection of PVL and methicillin resistance (mecA) genes. (A) Comparison of single target versus multiplex PCR using control strain CA05. Lane M, 1 Kb Plus DNA ladder (Invitrogen). (B) Optimized multiplex PCR assay showing control strains used for assay development. Lane 1, ATCC 51811; lane 2, ATCC 29213; lane 3, ATCC 25923; lane 4, CLS-1807; lane 5, ATCC 33591; lane 6, HIP 5827; lane 7, JCSC 4469; lane 8, N02-590; lane 9, CA05; lane 10, 8/6-3P; lane 11, MR108; lane 12, ATCC 14990; lane 14, PCR negative control; lanes M, 100-bp DNA ladder (New England BioLabs). Table 1 provides details for the strains.

VOL. 44, 2006 TABLE 2. Validation of multiplex PCR assay with well-characterized clinical control strains
Groupa Total no. of isolates tested Phenotypeb (OXA susceptibility) Single-target PCRc PVL mecA 16S rRNA Triplex PCRc PVL mecA

NOTES

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Correlation (%)

PVL PVL PVL PVL PVL PVL

a b

( ( ( ( ( (

) ) ) ) ) )

MSSA MSSA MRSA MRSA MS-CoNS MR-CoNS

30 13 30 45 30 30

S S R R S R

100 100 100 100 100 100

PVL ( ), PVL negative; PVL ( ), PVL positive. Oxacillin (OXA) susceptibility was determined with the VITEK system (bioMerieux) and by oxacillin susceptibility screening; S, susceptible; R, resistant (see Table 1 for details). c 16S rRNA, Staphylococcus genus specic; lukS/F-PV genes encode Panton-Valentin leukocidin S/F proteins; mecA, determinant of methicillin resistance; , positive for gene; , negative for gene.

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CFU per PCR for all the appropriate genes in each strain, which was quite compatible with the level of detection of single-target PCR assays (1 104 to 3 104 CFU per PCR). Clinical staphylococcal isolates were obtained from Calgary Laboratory Services (Alberta, Canada) and were characterized by phenotypic and genotypic methods as described previously (31). Multiplex PCR assay validation was performed by simultaneous comparison with the results of single-target PCR assays, as reported previously for PVL (15) and mecA (31), and with the antibiotic susceptibility phenotypes of 178 well-characterized clinical strains, including 30 PVL-negative MSSA strains, 13 PVL-positive MSSA strains, 30 PVL-negative MRSA strains, 45 PVL-positive MRSA strains, 30 PVL-negative methicillin-susceptible coagulase-negative staphylococci (MS-CoNS), and 30 PVL-negative methicillin-resistant CoNS (MR-CoNS) (Table 2). We found a 100% concordance among the results of these tests (Table 2). To address applicability and accuracy, we further applied our multiplex PCR assay to test a total of 287 MRSA, 280 MSSA, 220 MS-CoNS, and 228 MR-CoNS local clinical isolates randomly selected from our Calgary frozen isolate stock collection (the isolates were recovered from 1999 to 2003) and found PVL positivity rates of 1.9%, 2.1%, 0% and 0%, respectively, conrming the feasibility and practicality of our new assay. The low PVL positivity rates encountered with our local general S. aureus isolates are comparable to those encountered with isolates from other locales ( 5% of S. aureus isolates worldwide) (10, 12, 15, 28). Our new multiplex assay for the detection of PVL and methicillin resistance (mecA) genes represents a new tool to aid with the early identication of CA-MRSA strains. A rapidly increasing prevalence of serious CA-MRSA infections and deaths has been reported worldwide (7, 10, 11, 14, 17, 18), and a simple and rapid method of screening for the identication of S. aureus isolates carrying PVL genes is a crucial rst step in controlling the dissemination of this potentially virulent pathogen. Very recently, several groups have developed effective real-time PCR assays for the detection of the PVL genes, alone or in combination with the mecA, spa, or nuc gene (4, 13, 16, 21, 23). Due to the high costs of equipment and reagents, use of the real-time PCR technology has generally been limited to larger microbiology laboratories. To the best of our knowledge, our assay represents the rst conventional multiplex PCR assay capable of detecting both the PVL and the mecA

genes and was shown to be 100% accurate and reliable. Moreover, this assay is easily amenable to routine clinical use in any molecular biology laboratory with PCR capabilities. Our multiplex PCR assay was used in our regional MRSA surveillance program and helped identify and conrm the emergence of an outbreak of CA-MRSA infection in Calgary, Alberta, Canada in 2004, with the resultant expedient implementation of prevention and control measures (3, 8, 9).
We thank K. Hiramatsu and T. Ito for the kind gift of the control strains JCSC4469, CA05, 8/6-3P, MR108, and N315; F. Tenover for the gift of strain HIP 5827 (GISA); and M. Mulvey for the gift of strain N02-590. This work was partially supported by a CHR/CLS Research Grant (grant 17975) to K.Z. and an Operating Grant from the Centre for Antimicrobial Resistance (CAR), Calgary Health Region/CLS/University of Calgary. V.L. was a 2004 summer student.
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