A rapid, 2-well, multiplex real-time polymerase chain reaction assay for the detection of SCCmec types I to V in methicillin-resistant Staphylococcus aureus

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Diagnostic Microbiology and Infectious Disease 65 (2009) 384 – 391 www.elsevier.com/locate/diagmicrobio

A rapid, 2-well, multiplex real-time polymerase chain reaction assay for the detection of SCCmec types I to V in methicillin-resistant Staphylococcus aureus Håvard Valvatnea , Michelle I.A. Rijndersa , Ana Budimirb , Marie-Louise Boumansa , Albert J. de Neelingc , Patrick S. Beissera , Ellen E. Stobberingha , Ruud H. Deurenberga,⁎ a

Department of Medical Microbiology, Maastricht University Medical Center, Maastricht, The Netherlands b Department of Clinical and Molecular Microbiology, Clinical Hospital Centre Zagreb, Croatia c National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands Received 10 April 2009; accepted 11 August 2009

Abstract For us to assess the spread of methicillin-resistant Staphylococcus aureus (MRSA), typing of the staphylococcal cassette chromosome mec (SCCmec) is a valuable addition to existing typing methods, such as multilocus sequence typing (MLST). Traditional SCCmec typing assays, that is, that of Oliveira et al. and Ito et al., are polymerase chain reaction (PCR) based, requiring electrophoresis. We introduce a rapid, 2-well, multiplex real-time PCR assay that can be used directly on bacterial suspensions and is able to characterize SCCmec type I to V based on the detection of the ccr genes and the mec complex. The assay was evaluated on 212 clinical MRSA isolates from various countries, associated with MLST clonal complexes (CC) 1, 5, 8, 22, 30, and 45, as well as pig-associated CC398. When comparing the realtime PCR assay with traditional methods, the correct SCCmec element was identified in 209 (99%) of the 212 MRSA isolates. The new assay enables high-throughput analyses for SCCmec on large strain collections. © 2009 Elsevier Inc. All rights reserved. Keywords: MRSA; Real-time PCR; SCCmec

1. Introduction The genetic basis of β-lactam resistance in methicillinresistant Staphylococcus aureus (MRSA) is mecA, coding for the low-affinity penicillin-binding protein 2a. mecA is located on a genomic island designated staphylococcal cassette chromosome mec (SCCmec) (Ito et al., 1999). Currently, 8 main types of SCCmec (I–VIII) are distinguished (Daum et al., 2002; Ito et al., 2001, 2004; Okuma et al., 2002; Oliveira et al., 2006; Takano et al., 2008; Zhang et al., 2009). mecA is situated on a mec complex, 5 of which (A–E) are distinguished; of which A to C are

⁎ Corresponding author. Tel.: +31-43-3874644; fax: +31-43-3876643. E-mail address: [email protected] (R.H. Deurenberg). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2009.08.006

observed in MRSA. Class A (mecI–mecR1–mecA–IS431) is situated on SCCmec types II, III, and VIII; class B (IS1272–mecR1–mecA–IS431) on SCCmec types I, IV, and VI; and class C (IS431–mecR1–mecA–IS431) on SCCmec V and VII (Daum et al., 2002; Ito et al., 2001, 2004; Okuma et al., 2002; Oliveira et al., 2006; Takano et al., 2008; Zhang et al., 2009). In addition, SCCmec contains sitespecific cassette chromosome recombinases (ccr) responsible for the integration of SCCmec into the genome. The ccr genes exist in 2 forms, ccrAB and ccrC, and 4 main allotypes of ccrAB have been described. The ccr genes are designated ccrAB1 (SCCmec I), ccrAB2 (SCCmec II and IV), ccrAB3 (SCCmec III), ccrAB4 (SCCmec VI and VIII), and ccrC (SCCmec III, V and VII) (Daum et al., 2002; Ito et al., 2001, 2004; Okuma et al., 2002; Oliveira et al., 2006; Takano et al., 2008; Zhang et al., 2009). The regions that are not part of the mec complexes and ccr genes are called

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Table 1 Characteristics of the MRSA strains used in this study Lineagea

CC5

No. of strains

Country of origin

SCCmec type Ito et al./ Oliveira et al.

Real-time PCR

t001

1 4 1 11 3 1 1 1 2 1 1 1 1 2 1 1 2 6 2 1 1 5 1 3 2 1 7 3 1 2 1 1 11 1 3 6 6 2 1 12 9 1

Croatia Germany Belgium The Netherlands Belgium Croatia The Netherlands Germany Germany Germany Croatia Croatia Croatia The Netherlands Germany Croatia The Netherlands Belgium The Netherlands The Netherlands Croatia The Netherlands Belgium Germany The Netherlands Croatia Germany The Netherlands Croatia Germany The Netherlands Croatia The Netherlands The Netherlands Belgium Belgium Germany The Netherlands Croatia Germany The Netherlands Germany

I I I Ib I I I I I I I I I I I I I I I I I Ib I II II II II II II II II II II III IIIc IIIc III III III III III III

I I I I I I I I I I I I I I I I I I I I I I I II II II II II II II II II II III III III III III III III III III

t002 t041

CC8

t109 t143 t838 t2688 t2692 t2822 t008 t030 t051 t052

CC45 CC5

t303 t1605 t040 t4269 t002 t003

t041 t045

CC30 CC5

CC8

t2625 t012 t041 t045 t1107 t030 t037

CC45

Lineagea

spa type

t445

CC5

CC8

CC15 CC22 CC30 CC45

CC80

ST88 CC1 CC8

CC45 ST89 ST152 ST398

CC5

spa type

No. of strains

Country of origin

SCCmec type Ito et al./ Oliveira et al.

Real-time PCR

t001 t002

1 1 3 1 1 8 1 2 1 9 1 4 1 1 10 1 1 8 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2 2 2 2 1 4 1 2

Croatia Germany The Netherlands Belgium The Netherlands The Netherlands Croatia Croatia Germany The Netherlands Croatia The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands Croatia The Netherlands Belgium The Netherlands The Netherlands Croatia The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands The Netherlands Germany The Netherlands Germany The Netherlands

IV IV IV IV IV IV IV IV IV IV IV IV IV IV IVd IV IV IV IV IV IV IV IV IV IV V V V V V V V V V V V V V V V NTf NTg

IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV V V V V V V V Ve V V V V V Ve V V NTg

t045 t067 t447 t892 t008

t051 t068 t622 t346 t223 t253 t015 t038 t740 t1310 t044 t131 t786 t345 t588 t899 t1184 t1255 t1457 t2330 t2582 t3208 t445 t375 T355 t011 t034 t108 t002 t447

NT = not typeable. a Clonal complex (CC) or sequence type (ST) as determined by MLST. b From 10 isolates spa typed as t002 and 2 isolates spa typed as t040, the SCCmec element could not be characterized with the method of Oliveira and de Lencastre (2002) because no loci were detected. The method of Ito et al. (2001, 2004) and Okuma et al. (2002) characterized these SCCmec elements as type I. c From 2 isolates spa typed as t045 and all 6 isolates spa typed as t1107, the SCCmec element could not be characterized with the method of Oliveira and de Lencastre (2002) because only locus C (mecI) and D (dcs region) were detected. The method of Ito et al. (2001, 2004) and Okuma et al. (2002) characterized these SCCmec elements as type III. d From 5 isolates, the SCCmec element could not be characterized with the method of Oliveira and de Lencastre (2002) because no loci were detected. The method of Ito et al. (2001, 2004) and Okuma et al. (2002) characterized these SCCmec elements as type IV. e These SCCmec V elements lacked ORF V11 when tested with the real-time PCR assay. f This SCCmec element could not be characterized with the method of Oliveira and de Lencastre (2002) because only locus F (located between Tn554 and the chromosomal right junction [orfX]) was detected, and the method of Ito et al. (2001, 2004) and Okuma et al. (2002) detected no loci. g The method of Oliveira and de Lencastre (2002) characterized these elements as SCCmec IV. However, both the method of Ito et al. (2001, 2004) and Okuma et al. (2002) and the real-time PCR assay detected both ccrAB1 and ccrAB2.

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junkyard (J) regions. Each SCCmec element is divided into 3 J regions and SCCmec has the following general structure: J3–mec complex–J2–ccr genes–J1. Several

subtypes of SCCmec, which differ in the J regions compared with the main SCCmec elements, have been discovered (Chongtrakool et al., 2006; Kondo et al., 2007;

Table 2 Characteristics of the primers and probes used in the multiplex real-time PCR assay Locus

Primer/probe name

Sequence (5′→3′)

mecA

mecA forward mecA reverse mecA probe mecI forward mecI reverse mecI probe

TGAAGTGGTAAATGGTAATATCGACTTAA TAATTCGAGTGCTACTCTAGCAAAGAA CAAGCAATAGAATCATCAGATAA VIC CGTTATAAGTGTACGAATGGTTTTTGG GAATGGGAAGTTATGAATATCATTTGG CTTCTATTATATTATTCGCACTTGC VIC

0.6 0.6 4 0.9 0.9 4

ccrB1 forward ccrB1 reverse ccrB1 probe

AGGGACAAATTACACAAATAGAGCAA CCTGATATACCCCGATCTGCAT ATTGCCAATTTAACGGCTAT

FAM

0.9 0.9 4

ccrB2 forward ccrB2 reverse ccrB2 probe

GAAGGTTATAGTATCGACGGACAAATC ACCACGGTCAGCGTATATATCTTTAA CTGTGACTTCCATCATTT

FAM

0.6 0.9 4

ccrB3 forward ccrB3 reverse ccrB3 probe ccrC forward ccrC reverse ccrC probe IS1272 forward IS1272 reverse IS1272 probe

TCAGCTTATCTGAGCGTATGGAAGT GACCCATGAATACATTTTCGACAA ACAAGCAGTGGTAAATTGAT CCATCAAAATTGGGCTGTTCA GCTTACCTTTGACCAATATCACATCA CGAAGAAGTGGTAAAAGTGATAA GTGCAACTTCGTACCATCCTAAAA CGACCTGAGAATACAGATTGTGTGT TGTTAAAAGTGATTCTATATGCC

mecI

ccrB1

ccrB2

ccrB3

ccrC

IS1272

ORF V11

ORF V11 forward GGAGAGCAATTATTATGAGGGTAACAAA ORF V11 reverse TCTTCATCAGCCTCATTGATTGTTT ORF V11 probe TAGAATGTTGTCTCATGCAATT

5′ reporter dye

Reaction Prototype concentration strain name (μmol/L)

NED

0.6 0.6 4 0.9 0.9 4 0.8 0.9 8

NED

0.6 0.9 8

TET

TET

Accession no

SCCmec type

N315 Mu3 Mu50 JH1 JH9 MRSA252 HU25 85/3907 COL NCTC10442 9-62 9-34 9-33 N315 JCSC3063 M06/0075 RN7170 AR13.1/3330.2 Mu3 Mu50 JH1 JH9 MRSA252 MW2 USA300 USA300_TCH1516 JCSC4744 JCSC1978(8/6-3P) 80s-2 81/108(MR108) JCSC4788 JCSC4469 AR43/3330.1 M03-68 HGSA146 ANS46 AM904732 85/2082 JCSC3624(WIS) ANS46

BA000018 AP009324 BA000017 CP000736 CP000703 NC_002952 AF422694 AB047089 NC_002951 AY918259 AB033763 AY254742 AY254741 BA000018 AB127982 AM983545 AB261975 AJ810120 AP009324 BA000017 CP000736 CP000703 NC_002952 BA000033 NC_007793 CP000730 AB266531 AB063173 AB245471 AB096217 AB266532 AB097677 AJ810121 DQ106887 AY918287 AY918269 KM1381 AB037671 AB121219 AY918269

II II II II II II III III I I I I I II IIb IId II.4 IIe II II II II II IV IV IV IVa IVb IVc IVc IVc IVd IVe IVg IVh III III IIIb V III

COL NCTC10442 MW2 JCSC4744 JCSC1978(8/6-3P) 80s-2 AR43/3330.1 M03-68 JCSC3624(WIS)

CP000046 AB033763 BA000033 AB266531 AB063173 AB245471 AJ810121 DQ106887 AB121219

I I IV IVa IVb IVc IVe IVg V

See Donker et al. (2009)

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Kwon et al., 2005; Ma et al., 2002; Milheirico et al., 2007a; Oliveira & de Lencastre, 2002; Shore et al., 2005). Assignment of the SCCmec structure in MRSA is an important part in the characterization of MRSA clones. Moreover, SCCmec can be used as an additional marker besides spa typing, thereby increasing the discriminatory power of spa typing (Hallin et al., 2007; Strommenger et al., 2008). The SCCmec type can be determined with different polymerase chain reaction (PCR) assays. The most popular methods either detect mecA and different loci on SCCmec I to IV (Oliveira & de Lencastre, 2002) or determine the structure of the mec complex and the ccr genes (Ito et al., 2001; Okuma et al., 2002). Based on a recently proposed nomenclature (Chongtrakool et al., 2006), Kondo et al. (2007) have developed a PCR scheme, consisting of 2 multiplex PCRs for SCCmec typing and a further 3 multiplex PCRs for subtyping of the SCCmec element. The multiplex PCR assay described in 2002 by Oliveira & de Lencastre (2002) has recently been updated. This updated method includes the determination of the structure of SCCmec type V and VI, as well as improves the detection of SCCmec type I to IV (Milheirico et al., 2007b). Boye et al. (2007) and Zhang et al. (2005) have described a multiplex PCR assay, which mainly detects a single locus on SCCmec I to V. To type SCCmec I to V, we developed and evaluated a rapid multiplex real-time PCR assay that can be used directly on bacterial isolates in routine laboratories. Because of the technical limitations of real-time PCR assays in general, that is, the number of targets that can be detected (Klein, 2002), this assay determines only the ccr genes and the mec complex of the SCCmec element. 2. Materials and methods 2.1. Strain collection and genotyping COL, BK2464, ANS46, MW2, and WIS were used as reference strains for SCCmec I to V (Ito et al., 2004; Oliveira & de Lencastre, 2002). From our collection of MRSA strains, a random selection of 212 MRSA strains from individual patients from Belgium, Croatia, Germany, and the Netherlands representing different S. aureus lineages was used to validate the assay (Table 1). These isolates were identified as S. aureus by catalase and coagulase testing. The presence of mecA was determined by PCR (Donker et al., 2009). Spa typing was performed as described previously (Nulens et al., 2008), and the spa types were clustered into spa-CCs using the algorithm based upon repeat pattern (BURP) with Ridom StaphType 1.4 (Ridom GmbH, Munster, Germany) (Friedrich et al., 2006; Strommenger et al., 2006). Because it has been shown that spa typing/BURP results are in agreement with results obtained by multilocus sequence typing (MLST) (Strommenger et al., 2006, 2008), the associated MLST CCs were allocated through the SpaServer (Ridom, http://spaserver.ridom.de). The methods of Oliveira and de Lencastre (2002), Ito et al. (2001, 2004),

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Table 3 Multiplex real-time PCR loci detected on SCCmec types I to V SCCmec Multiplex real-time type PCR well 1

Multiplex real-time PCR well 2

mecA ccrB1 ccrB3 ORF V11 ccrB2 ccrC mecI IS1272 I II III IV V

+ + + + +

+ − − − −

− − + − −

− − − − +

− + − + −

− − + − +

− + + − −

+ − − + −

+ = present; − = absent.

and Okuma et al. (2002) were used to confirm the results obtained with the real-time PCR assay. Furthermore, PCRs for IS1272 and Tn554 were used to investigate the structure of SCCmec elements (Deurenberg et al., 2005). The combination of these methods enables the determination of the structure of SCCmec type I to V. 2.2. Multiplex real-time PCR The primers (Sigma-Genosys, Cambridge, UK) and TaqMan®-minor-groove-binding (MGB) probes (Applied Biosystems [ABI], Nieuwerkerk aan den IJssel, the Netherlands) used for the SCCmec multiplex real-time PCR were designed on the basis of available sequences using ClustalW and Primer Express 2.0 (ABI). The accession numbers for all sequences used are presented in Table 2. The multiplex realtime PCR assay was designed to amplify 2 regions on each SCCmec element and the mecA gene as an internal control for MRSA (Table 3). The ccrB genes of SCCmec type I to IV were aligned against each other to localize divergent regions allowing to design specific primers and probes for ccrB1, ccrB2, and ccrB3. In addition, ccrB2 sequences representing the subtypes of SCCmec types II and IV were used to find a probe that recognizes the subtypes of SCCmec types II and IV. Besides the ccrB genes, IS1272 and mecI were included in the real-time PCR assay to differentiate SCCmec type I and IV and SCCmec types II and III, respectively. The 1989bp open reading frame (ORF) V11 has been reported to be unique for SCCmec V (Ito et al., 2004; Zhang et al., 2005). Therefore, ORF V11 and ccrC were included in the real-time PCR assay to detect SCCmec type V (Table 3). The specificity of the primer and probe sequences was confirmed by screening sequence databases using BLAST (http://www. ncbi.nlm.nih.gov/blast). Possible interactions, such as crossbinding, hairpin loops, and homo- and heterodimer formation, between primer pairs and probes in the putative wells of the multiplex real-time PCR were analyzed using OligoAnalyzer 3.0 (Integrated DNA Technologies, Belgium). The final assay contained 8 primer and probe sets (Table 2). The multiplex real-time PCR assay comprised 2 wells of quadruple real-time PCRs per isolate. The first well contained primers and probes recognizing mecA, ccrB1, ccrB3, and ORF V11. The second well contained

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primers and probes for ccrB2, ccrC, mecI, and IS1272 (Table 3). The assay conditions, such as primer and probe concentrations, were optimized according to the guidelines from both the Primer Express 2.0 software package and the manual of the TaqMan Universal PCR Master Mix (ABI). The optimized assay was performed in a 96-well optical plate (ABI) with a final volume of 25 μL per reaction. All reactions consisted of 12.5-μL ABsolute™ QPCR ROX Mix (500 nmol/L) (ABgene, Epsom, UK), in addition to 7.5-μL primer and probe sets (Table 2). To each reaction, 5μL DNA (isolated from a bacterial suspension of 1.5 × 108 colony forming units [CFU]/mL using the Wizard Genomic DNA Purification Kit [Promega, Leiden, the Netherlands]) or bacterial suspension of 1.5 × 108 CFU/mL in water was added. Amplification and detection were performed with the ABI PRISM 7900HT Sequence Detection System (ABI) using the following program: 10 min at 95 °C, followed by 42 cycles of 15 s at 95 °C and 60 s at 60 °C. A reaction was considered positive when fluorescence levels exceeded the detection threshold before cycle 30. Amplification plots for the different SCCmec types are shown in Fig. 1. The results of the real-time PCR assay were assessed according to Table 3.

3. Results and discussion 3.1. Optimization of the SCCmec real-time PCR assay DNA, isolated from the reference strains for SCCmec type I to V, at concentrations between 2.5 × 10−3 and 2.5 × 10−6 μg/μL, was tested in a matrix of different concentrations of forward primer (0.3–0.9 μmol/L), reverse primer (0.3–0.9 μmol/L), and probe (1–8 μmol/L). Ct values ranging from 20 to 25 were obtained for each real-time PCR target, which was considered optimal for the assay (data not shown). The optimal concentration for the primers and probes (ΔR values higher than 1.0) are presented in Table 2. The optimal DNA concentration was 2.5 × 10−5 μg/μL. Because the PCR conditions were identical for all primers and probes, the 8 real-time PCRs were combined in 2 multiplex assays. No major differences in the amplification curves for the targets were observed in the single and multiplex PCRs. Typical amplification plots for the SCCmec type I to V in the multiplex real-time PCR assay are shown in Fig. 1. Next, the optimal primer and probe concentrations combined in multiplex real-time PCRs were tested with a heated bacterial suspension of 1.5 × 108 CFU/mL in water. No changes in Ct values and signals were observed between the purified DNA and the heated bacterial suspensions (data

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not shown). Therefore, all further tests were performed on bacterial suspensions. 3.2. Comparison of the real-time PCR with established SCCmec typing methods The method of Oliveira and de Lencastre (2002) and the method of Ito et al. (2001, 2004) and Okuma et al. (2002) gave consistent results when typing the SCCmec element from 161 (76%) of the 212 MRSA strains: 39 SCCmec type I, 32 SCCmec type II, 33 SCCmec type III, and 57 SCCmec type IV elements (Table 1). From the remainder 51 MRSA strains, the SCCmec element from 48 strains, that is, 12 SCCmec type I, 8 SCCmec type III, 5 type SCCmec IV, and 23 SCCmec type V elements, could only be characterized with the method of Ito et al. (Table 1). It has been shown previously that variants of SCCmec type I to V cannot be typed with the method of Oliveira et al. but could only be characterized with the method of Ito et al. (Kim et al., 2007; Shore et al., 2005). It is known that SCCmec type V cannot be detected with the method of Oliveira et al. but can only be detected with the method of Ito et al., as well as with several other methods (Boye et al., 2007; Zhang et al., 2005). The SCCmec elements from 3 MRSA strains could not be typed using both the method of Oliveira et al. and Ito et al. (Table 1). The real-time PCR was able to characterize all 209 SCCmec elements that could be characterized with the method of Oliveira et al. and/or Ito et al. From the 3 SCCmec elements that could not be typed with these methods, 1 was characterized as SCCmec type V (positive for ccrC, mecA, and ORF V11 and negative for ccrAB1, ccrAB2, ccrAB3, IS1272, and mecI) and 2 SCCmec elements were not typeable with the real-time PCR (Table 1). 3.3. Unanticipated real-time PCR results Although variants of SCCmec type I to V, as well as novel SCCmec elements, have been described previously in many studies (Chongtrakool et al., 2006; Kondo et al., 2007; Kwon et al., 2005; Ma et al., 2002; Milheirico et al., 2007a; Oliveira & de Lencastre, 2002; Shore et al., 2005), some unexpected results with the real-time PCR assay were obtained, which could complicate the data analyses. SCCmec type III elements harbor both ccrB3 and mecI (Ito et al., 2001). In addition, ccrC is present in most of these elements, but this is not a prerequisite for defining SCCmec type III, because not all SCCmec type III elements carry the mercury resistance locus, which includes the ccrC gene (Chongtrakool et al., 2006; Ito et al., 2004). The realtime PCR assay failed to detect ccrC in 10 (24%) of the 41 SCCmec type III elements. Aligning the available ccrC sequences, it was observed that the homology of ccrC was less than 90% for SCCmec III and V. The primer and probe set

Fig. 1. Typical amplification plots of reference strains for SCCmec type I to V. Graphs for each fluorescent dye (TaqMan®-MGB probe) are shown. (A) SCCmec type I, positive for mecA, ccrB1 in well 1 and IS1272 in well 2. (B) SCCmec type II, positive for mecA in well 1 and ccrB2 and mecI in well 2. (C) SCCmec type III, positive for mecA, ccrB3 in well 1 and ccrC and mecI in well 2. (D) SCCmec type IV, positive for mecA in well 1 and ccrB2 and IS1272 in well 2. (E) SCCmec type V, positive for mecA and ORF V11 in well 1 and ccrC in well 2.

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for ccrC was designed for ccrC in SCCmec type V and will, therefore, not always be positive for SCCmec type III. ORF V11 was not detected in 2 (9%) of the 23 SCCmec type V elements. Both elements were characterized as SCCmec type V with the method of Ito et al. (2004). Being negative for ccrB3 and mecI and positive for mecA and ccrC, it was concluded that these 2 elements were SCCmec type V, although they lack ORF V11 in the real-time PCR assay, indicating that not all SCCmec type V elements contain ORF V11. The 2 SCCmec elements that were not typeable with the real-time PCR harbored not only ccrB2 and IS1272 but also ccrB1, which makes it impossible to define the correct SCCmec type. These elements were incorrectly typed as SCCmec IV using the method of Oliveira and de Lencastre (2002). Other studies have shown that isolates can contain multiple ccr genes (Berglund et al., 2008; Heusser et al., 2007; Qi et al., 2005; Takano et al., 2008). An SCCmec element containing both ccrB1 and ccrB2 has been observed recently in Switzerland (Ender et al., 2009). 3.4. Conclusion A multiplex real-time PCR assay was developed for the rapid detection of SCCmec type I to V based on de characterization of the ccr genes and the mec complex. The real-time PCR assay generated easily discriminated signals for SCCmec type I to V directly from bacterial colonies only 1.5 h after the real-time PCR assay was started. For the other SCCmec typing methods (Ito et al., 2001, 2004; Okuma et al., 2002; Oliveira & de Lencastre, 2002), it is necessary to isolate the DNA from MRSA, and the assay, including a conventional PCR assay and gel electrophoresis, takes approximately 4 h. Furthermore, the real-time PCR assay is suitable for high-throughput analyses (Klein, 2002). A limitation of the real-time PCR is that it is more expensive compared with a conventional PCR assay ($2 versus $0.2). A further limitation of the real-time PCR assay is that it does not detect the subtypes of the major SCCmec elements and the rarely observed SCCmec VI to VIII (Oliveira et al., 2006; Takano et al., 2008; Zhang et al., 2009). However, future real-time PCR technology should make it possible 1) to detect more loci on SCCmec type I to V to improve the discriminatory power of the real-time PCR assay, 2) to detect the subtypes of the major SCCmec elements, and 3) to detect the rarely observed SCCmec type VI to VIII, as well as other novel SCCmec elements, not yet described (Klein, 2002).

Acknowledgments The authors thank H. de Lencastre from The Rockefeller University, New York, NY, for providing the reference strains for SCCmec I to IV and T. Ito from the Juntendo University, Tokyo, Japan, for providing the reference strain for SCCmec V. They thank Selma Herngreen for sharing her expertise on multiplex real-time PCR.

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