Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions

Abstract

Staphylococcal cassette chromosome mec (SCCmec) typing, in combination with genotyping of the Staphylococcus aureus chromosome, has become essential for defining methicillin-resistant S. aureus (MRSA) clones in epidemiological studies. We have developed a convenient system for SCCmec type assignment. The system consists of six multiplex PCRs (M-PCRs) for identifying the ccr gene complex (ccr), the mec gene complex (mec), and specific structures in the junkyard (J) regions: M-PCR with primer set 1 (M-PCR 1) identified five types of ccr genes; M-PCR 2 identified class A to class C mec; M-PCRs 3 and 4 identified specific open reading frames in the J1 regions of type I and IV and of type II, III, and V SCCmec elements, respectively; M-PCR 5 identified the transposons Tn554 and PsiTn554 integrated into the J2 regions of type II and III SCCmec elements; and M-PCR 6 identified plasmids pT181 and pUB110 integrated into J3 regions. The system was validated with 99 MRSA strains carrying SCCmec elements of different types. The SCCmec types of 93 out of the 99 MRSA strains could be assigned. The SCCmec type assignments were identical to those made with a PCR system that uses numerous primer pairs to identify genes or gene alleles. Our system of six M-PCRs is thus a convenient and reliable method for typing SCCmec elements.

FIG. 1.

Schematic structures of representative SCCmec elements based on the nucleotide sequences deposited in the EMBL/GenBank/DDBJ database under the accession numbers listed in Table 1. Circles indicate the ccr genes that can be identified by M-PCR 1. The mec gene complexes that can be identified by M-PCR 2 are indicated by squares. Black bars indicate the locations of the J1 region-specific primers used for M-PCRs 3 and 4. The locations of primers used for the amplification of the entire type II.4 SCCmec region of strain RN7170 are indicated by red arrowheads.

FIG. 2.

Six multiplex PCRs for SCCmec type assignment. (A) M-PCR 1 for identification of ccr genes for the assignment of the type of ccr gene complex. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, N315; lane 3, 85/2082; lane 4, CA05; lane 5, WIS; lane 6, HDE288. DNA fragments of the expected sizes for each ccr gene were amplified. The DNA fragment corresponding to mecA served as an internal control in each lane. (B) M-PCR 2 for identification of three gene alleles for assignment of the mec gene complex. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, N315; lane 3, 85/2082; lane 4, CA05; lane 5, WIS. DNA fragments of the expected sizes for class A mec (lanes 2 and 3), class B mec (lanes 1 and 4), and class C mec (lane 5) were amplified. (C) M-PCR 3 for J1 region difference-based subtyping of type I and type IV SCCmec elements, which carry the class B mec. Chromosomal DNAs from standard strains were used as templates. Lane 1, NCTC10442; lane 2, CA05; lane 3, 8/6-3P; lane 4, 81/108; lane 5, JCSC4469. DNA fragments of the expected sizes for each J1 region were amplified. (D) M-PCR 4 for J1 region-based subtyping of type II and type III SCCmec elements, which carry the class A mec, and the type V SCCmec, which carries the class C mec. Chromosomal DNAs from standard strains were used as templates. Lane 1, N315; lane 2, JCSC3063; lane 3, BK351; lane 4, RN7170; lane 5, 85/2082; lane 6, WIS. (E) Identification of resistance determinants. Transposons (Tn554 and ΨTn554) were assigned by M-PCR 5 (lanes 1 to 3), and plasmids (pUB110 and pT181) were assigned by M-PCR 6 (lanes 4 and 5). SCCmercury was assigned with an M-PCR with the four sets of primers listed in Table 2 (lane 6). Chromosomal DNAs from standard strains were used as templates. Lanes 1 and 4, N315; lane 2, BK645; lanes 3, 5, 6, and 85/2082. MWM, molecular weight marker.