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Antimicrobial Agents and Chemotherapy, September 2007, p. 3374-3377, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00275-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Update to the Multiplex PCR Strategy for Assignment of mec Element Types in Staphylococcus aureus{triangledown}

Catarina Milheiriço,1 Duarte C. Oliveira,1* and Hermínia de Lencastre1,2

Laboratory of Molecular Genetics, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal,1 Laboratory of Microbiology, The Rockefeller University, New York, New York 100212

Received 23 February 2007/ Returned for modification 7 April 2007/ Accepted 10 June 2007


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ABSTRACT
 
Staphylococcal cassette chromosome mec (SCCmec) typing is important for the identification and definition of methicillin-resistant Staphylococcus aureus clones, and for routine purposes, multiplex PCR assays are the most adequate for SCCmec typing. Here, we describe an update to the multiplex PCR strategy for SCCmec typing that we described in 2002 so that SCCmec types IV and V may be properly identified.


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TEXT
 
Methicillin-resistant Staphylococcus aureus (MRSA) strains are characterized by the presence of a large heterologous mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec), which includes the mecA gene, the central element of methicillin resistance (4). Besides the mec gene complex (which comprises the mecA gene and its regulators, mecI and mecR1), SCCmec contains the ccr gene complex, which encodes recombinases responsible for the mobility of SCCmec (7). Several SCCmec types have been defined by use of the combination of the class of the mec gene complex and the ccr allotype (1, 3-5, 9, 16). The remaining parts of SCCmec are called J regions (regions J1, J2, and J3), which constitute nonessential components of the cassette, although in some cases these regions carry additional antibiotic resistance determinants. J1 is the region between the chromosomal left junction and the ccr complex, J2 is the region between the ccr complex and the mec complex, and J3 is the region between the mec complex and the chromosomal right junction. Therefore, the structural organization of SCCmec may be summarized as J1-ccr-J2-mec-J3. Variations in the J regions within the same mec-ccr combination are used to define SCCmec subtypes.

In 2002, we described a multiplex PCR strategy for the rapid assignment of SCCmec types to MRSA strains. That strategy was able to properly identify SCCmec types I to III and some epidemiologically relevant variants (e.g., subtypes IA and IIIA) by probing eight loci scattered through the mec elements and generating specific amplification fragments of three to five bands (13). SCCmec type IV, which at that time was not yet recognized as an important structural type mainly due to its spread among community-acquired MRSA (CA-MRSA) strains, was not properly identified since it was positive only for the internal positive control and the dcs locus, which is also present in SCCmec types I and II. Here, we report an update to the previously described "SCCmec multiplex PCR strategy" in order to better characterize SCCmec type IV and also to include the detection of the recently described SCCmec type V.

Table 1 lists the characteristics of the primers used for the updated version of the SCCmec multiplex PCR. In order to minimize the complexity of the multiplex PCR, the detection of linearized plasmids pUB110 and pT181 was abandoned. These loci are not critical for SCCmec type assignment, and its utility was to discriminate subtypes IA (positive for pUB110) and IIIA (negative for pT181). Eight new primers were added for the detection of ccrB allotype 2 (specific for SCCmec types II and IV), ccrC (specific for SCCmec type V), the SCCmec type III J1 region, and the SCCmec type V J1 region.


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  		TABLE 1. Primers used in the updated version of SCCmec multiplex PCR

The conditions for the multiplex PCR assay were first optimized by using the following prototype strains: COL, SCCmec type I (16); N315, SCCmec type II (4); ANS46, SCCmec type III (16); MW2, SCCmec type IVa (1); 8/6-3P, SCCmec type IVb (9); Q2314, SCCmec type IVc (6); JCSC4469, SCCmec type IVd; AR43/3330.1, SCCmec type IVE (17); M03-68, SCCmec type IVg (8); HAR22, SCCmec type IVh (11); WIS, SCCmec type V (5); and HDE288, SCCmec type VI (15). For validation purposes, a diverse collection of 60 MRSA isolates previously characterized in terms of their genetic backgrounds and SCCmec types was tested by use of the updated SCCmec multiplex PCR assay (Table 2). All assays were performed in a T1 thermocycler (Biometra, Germany). The optimal cycling conditions were the following: 94°C for 4 min; 30 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 4 min. Each PCR mixture, in a final volume of 50 µl, contained 5 ng of chromosomal template; 1x PCR buffer with 1.5 mM MgCl2 (Applied Biosystems); 40 µM (each) deoxynucleoside triphosphate (MBI Fermentas, Hanover, MD); 0.2 µM primers kdp F1 and kdp R1; 0.4 µM primers CIF2 F2, CIF2 R2, RIF5 F10, RIF5 R13, SCCmec III J1F, SCCmec III J1R, SCCmec V J1 F, and SCCmec V J1 R; 0.8 µM primers mecI P2, mecI P3, dcs F2, dcs R1, mecA P4, mecA P7, ccrB2 F2, ccrB2 R2, ccrC F2, and ccrC R2; and 1.25 U of Amplitaq DNA polymerase (Applied Biosystems). The PCR products (10 µl) were resolved in a 3% Seakem LE (Cambrex, Rockland, ME) agarose gel in 0.5% Tris-borate-EDTA buffer (Bio-Rad, Hercules, CA) at 4 V/cm for 2.5 h and were visualized with ethidium bromide.


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  		TABLE 2. Prototype strains and representative collection used for validation of updated SCCmec multiplex PCR assay

Figure 1 illustrates the amplification patterns obtained for the prototype strains. SCCmec types I to III generate specific amplification patterns of three to five bands. SCCmec type IV generates an amplification pattern of three bands (mecA, ccrB2, and dcs) for subtypes a to d, g, and h; and subtypes IVE and IVF generate only two bands, since they are negative for the dcs locus. SCCmec type V also originates a pattern of three bands (mecA, ccrC, and J1). SCCmec type VI, which appears to be an "exotic" local variant (14), generates only two bands (mecA and dcs), and its assignment must be confirmed by ccrAB allotyping (ccrAB4). Note that SCCmec types IV and VI previously could not be discriminated since both were positive only for mecA and dcs.


Figure 1
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  		FIG. 1. Amplification patterns for the prototype strains obtained with the updated SCCmec multiplex PCR strategy. Lane 1, SCCmec type I (strain COL); lane 2, type II (N315); lane 3, type III (ANS46), lane 4, type IVa (MW2); lane 5, type IVb (8/6-3P); lane 6, type IVc (Q2314); lane 7, type IVd (JCSC4469); lane 8, type IVE (AR43/3330.1); lane 9, type IVg (M03-68); lane 10, type IVh (HAR22); lane 11, type V (WIS); lane 12, type VI (HDE288); lanes M, DNA molecular size marker (1-kb DNA Ladder Plus; Invitrogen Life Technologies, Carlsbad, CA).

As illustrated in Table 2, the updated SCCmec multiplex PCR assay performed well in assigning SCCmec types to the 60 diverse MRSA isolates. As expected, subtypes IA and IIIA, defined by the presence of pUB110 and the absence of pT181, respectively, could not be detected, whereas the sporadically occurring subtype IIIB, defined as being negative for the J3 region specific locus, was still detected. Strains PL72 and POL3 were incorrectly assigned to SCCmec type VI. These sporadically occurring strains were first tentatively classified as SCCmec type IV (13) and after ccrB sequencing (14) were classified as new type I variants with a partial deletion within the J1 region, which includes the J1-specific locus of SCCmec type I included in the multiplex PCR assay. This unique example of an incorrect assignment reinforces the importance of confirming multiplex PCR results by ccr gene complex typing, either by conventional PCR allotyping (12) or by DNA sequencing (15), at least for a subset of strains belonging to the same clonal type or whenever a new clone is detected.

In short, the updated version of the previously described SCCmec multiplex PCR strategy enables the rapid presumptive assignment of all known SCCmec types to MRSA strains. Whereas the previous version enabled the prompt identification of only SCCmec types I to III, since SCCmec type IV was poorly identified and SCCmec type V was not detected, the means for the proper identification of SCCmec types IV and V was added to this updated version. The prompt detection of SCCmec types IV and V is particularly relevant for the characterization of the recent threat of CA-MRSA, mostly characterized by these two structural variants of the mec element. In practical terms, changes to the previous protocol were kept to a minimum, and so the implementation of this SCCmec typing strategy will be straightforward, particularly for those laboratories currently using the previously described SCCmec multiplex PCR strategy.

The importance of SCCmec typing is well illustrated by the fact that the proposal by Enright and colleagues (2) that MRSA clones be named according to their multilocus sequence types and SCCmec types (e.g., clone ST5-MRSA-II), which was agreed to by a subcommittee of the International Union of Microbiology Societies in Tokyo, Japan, in 2002, is currently consensual in the specialized literature. In this context, rapid and easy assays for the detection of SCCmec types, such as the multiplex PCR typing strategy described in this study, are critical tools for the proper characterization of MRSA clones. The SCCmec element, which carries the determinant for "broad-spectrum" beta-lactam resistance in staphylococci, is a critical epidemiological marker for MRSA clones. However, besides being an important tool for surveillance studies, SCCmec typing of large international collections of isolates has contributed dramatically to the elucidation of the origin(s) and evolutionary history of contemporary MRSA clones.


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ACKNOWLEDGMENTS
 
We thank T. Ito, D. C. Coleman, R. Daum, K. T. Park, W. B. Grubb, J. Etienne, and A. Tomasz for having kindly given some of the prototype and reference strains used in this study.

Partial support for this study was provided by projects POCI/BIA-MIC/58416/2004 from the Fundação para a Ciência e Tecnologia (FCT), Lisbon, Portugal, and FCG-55068 from the Fundação Calouste Gulbenkian, Lisbon, Portugal, both awarded to H. de Lencastre. C. Milheiriço and D. C. Oliveira were supported by grants SFRH/BD/23010/2005 and SFRH/BPD/9374/2002, respectively, from FCT.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal. Phone: (351) 21 446 9862. Fax: (351) 21 442 8766. E-mail: dco{at}itqb.unl.pt Back

{triangledown} Published ahead of print on 18 June 2007. Back


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Antimicrobial Agents and Chemotherapy, September 2007, p. 3374-3377, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00275-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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