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Antimicrob Agents Chemother. 2006 Mar; 50(3): 1001–1012.
PMCID: PMC1426434

Staphylococcal Cassette Chromosome mec (SCCmec) Typing of Methicillin-Resistant Staphylococcus aureus Strains Isolated in 11 Asian Countries: a Proposal for a New Nomenclature for SCCmec Elements

Abstract

A description of staphylococcal cassette chromosome mec (SCCmec) elements carried by 615 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 11 Asian countries is reported, and a novel nomenclatural system based on their structures is proposed. The 615 strains were classified as type 3A (370 strains), type 2A (207 strains), type 2B (32 strains), type 1B (1 strain), and nontypeable (5 strains). The previously reported type III SCCmec (DDBJ/EMBL/GenBank accession no. AB037671) carried by the MRSA strain 85/2082 was ascertained to be composed of two SCC elements, type 3A SCCmec and SCCmercury. PCR analysis indicated that 310 of 370 type 3A SCCmec strains carried both SCC elements. These strains were prevalent in eight countries: Thailand, Sri Lanka, Indonesia, Vietnam, Philippines, Saudi Arabia, India, and Singapore. The remaining 60 type 3A SCCmec strains differed with respect to the left extremity polymorphism or to the presence of ccrC. Among these, two were identified as carrying only type 3A SCCmec elements, but their left extremities differed. Type 2A SCCmec strains predominated in Korea and Japan, although the frequency of the presence of ant(4′)-1 gene downstream of mecA varied (53% for Korean strains; 93% for Japanese strains). Various SCCmec elements were identified in the tested strains, and limited numbers were identified by their multilocus sequence typing genotypes. These data suggest that numerous MRSA clones are disseminated in Asian hospitals, and these consist of minor clones that are presumed to have arisen locally and major clones that are presumed to have been introduced from other countries.

Methicillin, a semisynthetic penicillin that is poorly hydrolyzed by penicillinase, was first used clinically in 1960. Only 1 year later, Staphylococcus aureus strains that showed resistance to methicillin were reported (17). Since then, methicillin-resistant Staphylococcus aureus (MRSA) strains have appeared in countries worldwide and continue to be one of the most common hospital pathogens (1). It has been shown that methicillin-susceptible S. aureus (MSSA) strains become MRSA strains by the acquisition of a staphylococcal cassette chromosome mec (SCCmec) element carrying the mecA gene, which is responsible for methicillin resistance (16, 18). SCCmec elements, unique genomic islands that are found in staphylococcal species, have two essential components, the ccr gene complex (ccr) and the mec gene complex (mec) (10, 13). The ccr gene complex is composed of ccr genes and surrounding open reading frames (ORFs), and the mec gene complex is composed of the mecA gene, regulatory genes, and insertion sequences upstream or downstream of mecA. Several mec and ccr allotypes have been found among SCCmec elements, which has led to the following classification (14, 15, 22): type I SCCmec, carrying class B mec and type 1 ccr; type II SCCmec, with class A mec and type 2 ccr; type III SCCmec, with class A mec and type 3 ccr; type IV SCCmec, with class B mec and type 2 ccr; and type V SCCmec, class C2 mec and type 5 ccr.

Recently, MRSA has become increasingly prevalent in community-acquired infections (3, 6, 7, 28, 37). SCCmec typing and genotyping have provided strong evidence for the independent origins of health care-associated MRSA and community-acquired MRSA (C-MRSA) (2, 5, 21, 25, 26, 29, 33, 39). While SCCmec typing has become essential for the characterization of MRSA clones in epidemiological studies, there are two problematic issues. One concerns assigning of SCCmec elements, and the other concerns naming novel elements or variants. We have assigned types of SCCmec elements by determining mec classes and ccr types. On the other hand, Oliveira and Lencastre have developed a multiplex PCR technique for assigning SCCmec elements based on the identification of eight loci (A to H) mostly located in the J regions (31). However, we think that the concept that SCCmec types can be assigned by determining mec class and ccr type first has been agreed upon by staphylococcal researchers. Nonetheless, the nomenclature of SCCmec types remains problematic. Historically, two type IV SCCmec elements have been described. In 2005, two new type II SCCmec elements (IIb and IIB) were reported (12, 36). Rules for naming SCCmec elements that are acceptable to all researchers need to be established. We have developed a new nomenclature for SCCmec types, which is an attempt to resolve confusion in the field and to facilitate the identification of novel SCCmec elements. This nomenclature is based on the structure of SCCmec elements, and it is suitable for describing previously reported SCCmec elements as well as those distributed in Asian countries, as far as we can determine. Here, we report on the characteristics of Asian MRSA clones. Our data suggest that MRSA clones may have been generated by the integration of SCCmec elements into MSSA clones that have propagated in each country and that MRSA clones in Asian hospitals are more divergent than previously reported (8, 19).

MATERIALS AND METHODS

Bacterial strains.

A total of 615 methicillin-resistant Staphylococcus aureus strains were analyzed in this study. Specimens were obtained from health care facilities in the following countries from 1998 to 1999 by the Asian Network for Surveillance of Resistant Pathogens: China (Beijing Children's Hospital, Beijing, and Shanghai Children's Hospital, Shanghai), 40 strains; India (Christian Medical College, Vellore), 37 strains; Indonesia (University of Indonesia, Jakarta), 60 strains; Japan, 138 strains; Korea (Samsung Medical Center, Seoul), 105 strains; Philippines (Research Institute for Tropical Medicine, Manila), 15 strains; Saudi Arabia (King Saud University Hospital, Riyadh), 19 strains; Singapore (National University of Singapore, Singapore), 87 strains; Sri Lanka (University of Colombo, Colombo), 29 strains; Thailand (Mahidol University, Bangkok), 49 strains; and Vietnam (University of Medicine and Pharmacy, Ho Chi Minh City), 36 strains.

All strains collected by the Asian Network for Surveillance of Resistant Pathogens program (other than mecA-negative strains) were tested, and some were tested by Ko et al. previously (19). S. aureus ATCC 25923 was used as a quality control for susceptibility testing.

Five MRSA strains, NCTC10442, N315, 85/2082, CA05, and WIS (WGB8318), were used for the standard strains of type 1A (type I), type 2A (type II), type 3A (type III), type 2B (type IV), and type 5C (type V) SCCmec elements.

PCR-based assignment of SCCmec elements.

Chromosomal DNA was extracted from MRSA strains by the small-scale phenol extraction method and used as templates. PCRs were performed using a thermal cycler Gene Amp 9600 (Perkin-Elmer Cetus Instruments, Emeryville, CA). Reaction mixtures contained 10 ng template DNA, oligonucleotide primers (0.2 μM), 400 μM each deoxynucleoside triphosphate, 1× Ex Taq buffer with magnesium, and 2 U Ex Taq polymerase (Takara Bio, Inc., Kyoto, Japan) in a final volume of 50 μl. PCRs consisted of 30 cycles of denaturation (95°C, 30 s), annealing (50°C, 60 s), and extension (72°C, 2 min). Four microliters of each reaction mixture was subjected to agarose gel electrophoresis to detect amplified DNA fragments. The primer sets used for the assignment of SCCmec elements are listed in Table Table1.1. The presence of the mecA gene was first checked to confirm MRSA status. Class A mec strains were identified by amplification of a DNA fragment with one primer specific for the mecI gene (mI4) and the other specific for the mecR1 gene (mcR5). Similarly, class B mec and class C mec strains were identified by amplification of fragments with primers specific for mecA (mA6) and IS1272 (IS5) or for IS431 (IS2) and mecA (mA2), respectively. The ccr gene complexes were typed by multiplex PCR using four primers: βc, a degenerate inosine-containing primer that recognizes ccrB1, ccrB2, and ccrB3; α1, a primer specific for ccrA1; α2, a primer specific for ccrA2; and α3, a primer specific for ccrA3. The ccr gene type was determined by the size of the amplified DNA fragment as follows: type 1 ccr, 0.7 kb; type 2 ccr, 1 kb; and type 3 ccr, 1.6 kb (Table (Table1).1). Strains for which DNA fragments were not amplified with the above primers were examined for the presence of type 4 ccr.

TABLE 1.
List of primers used in this study

The J region in the SCCmec element was further classified into three subregions, J1 (formerly called the L-C region), J2 (the former C-M region), and J3 (the former M-R region). Table Table11 shows the list of primers used to distinguish J regions.

PCR-based identification of small type 3A SCCmec elements.

DNA fragments encompassing the entire SCCmec elements of strains JCSC290 and JCSC1716 were amplified by long-range PCR using the Expand High Fidelity PCR system following the manufacturer's recommendations (Roche Diagnostics Co., Indianapolis, IN). Briefly, reactions were performed in a final volume of 50 μl and contained 10 ng template DNA, oligonucleotide primers (0.3 μM), 200 μl each deoxynucleoside triphosphate, 1× Expand High Fidelity buffer, 1.5 mM MgCl2, and 2.6 U Expand High Fidelity PCR system enzyme mix. PCR consisted of the following: a denaturation step (94°C, 2 min); 10 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 8 min); 20 cycles of denaturation (94°C, 15 s), annealing (50°C, 30 s), and extension (68°C, 12 min); and a final elongation step (72°C, 7 min).

The primer sets used for amplifying the entire SCCmec region of strain JCSC290 and amplicon sizes are as follows: for the region from orfX to pT181 insert, cR1 and pT181-1 (6.3 kb); for the region from pT181 to mecA, pT181-2 and mA3 (7.2 kb); for the region from mecA to integrated ΨTn554 (cad), mA4 and cad3 (8.8 kb); for the region flanking ΨTn554 (cad), mN5 and mN23 (7.0 kb); for the region from ΨTn554 (cad) to ccr, cad2, and β4 (7.2 kb); and for the region from ccr to chromosomal sequence flanking the left end of SCCmec, β3 and cLt3 (6.7 kb). DNA fragments encompassing the entire SCCmec region of JCSC1716 were amplified by long-range PCR with the same primer sets used for JCSC290 except for the following two sets: for the region from orfX to the IS431 insert, cR1 and IS431mec (3.2 kb); for the region from orfX to the mecA gene, cR1 and mA3 (6.2 kb), mA4 and cad3 (8.7 kb), mN5 and mN23 (6. 8 kb), cad2 and β4 (7.4 kb), and β3 and cLt3 (6.7 kb). The locations of primers are indicated in Fig. Fig.1.1. PCR products were purified with the QIA quick PCR purification kit (QIAGEN, Hilden, Germany) and sized by agarose gel electrophoresis.

FIG. 1.
Structural comparison of SCCmec elements. The structures of SCCmec elements are based on the nucleotide sequences deposited in the DDBJ/EMBL/GenBank databases under accession nos. ...

PCR detection of spontaneous excision of the type 3A SCCmec and SCCmercury elements.

Five primer sets were used to detect the precise excision of SCCmec and SCCmercury in strains 85/2082, JCSC290, and JCSC1716 by standard PCR using the same conditions as for SCCmec typing. The locations of primers are shown in Fig. Fig.2,2, and their sequences are listed in Table Table1.1. For strain 85/2082, primer pairs cR1 and cLt4, cR1 and mN19, and mN17 and cLt5 were used to detect the precise excision of type 3A SCCmec-SCCmercury, SCCmercury, and type 3A SCCmec, respectively. Primers cR1and cLt5 were used to detect attB (350 bp) in JCSC290 and JCSC1716.

FIG. 2.
Identification of small type 3A SCCmec elements. The structures of three type 3A SCCmec elements and characteristic nucleotides at both extremities are illustrated. (A) The structure of the type 3A SCCmec element and of the SCCmercury element (formerly ...

Nested PCR was also carried out to detect the precise excision of SCCmec and SCCmercury following the procedure described previously (16).

Three sets of primer pairs were used: set A (the outer primers, cLt2 and cR2, and the inner primers, cLt5 and cR1); set B (the outer primers, cLt2 and mN20, and the inner primers, cLt5 and mN17); set C (the outer primers, cR2 and mN19, and the inner primers, mN18 and cR1). Set A was used to detect attB (350 bp) generated by the excision of type 3A SCCmec from strains JCSC290 and JCSC1716 or by the excision of a composite of type 3A SCCmec and SCCmercury from strain 85/2082. Set B and set C were used to detect the excision of type 3A SCCmec and SCCmercury, respectively, from 85/2082.

Multilocus sequence typing (MLST).

MLST was performed as previously described (9). Alleles of the seven loci were assigned by comparing the sequences of each locus to those in the S. aureus MLST database (www.mlst.net). Sequence types were determined according to the pattern of combination of the seven alleles, and clonal complexes were defined by the BURST (based upon related sequence types) program available on the MLST website.

RESULTS AND DISCUSSION

A proposal for SCCmec element nomenclature.

We propose a new nomenclatural system for SCCmec elements that has three features. The first feature is a description of SCCmec type. It appears to be widely accepted that SCCmec types are defined by ccr type and mec class. An SCCmec element can be simply described by ccr type (indicated by a number) and mec class (indicated by an uppercase letter). Alternatively, roman numeral designations could be used instead of combinations of ccr and mec as indicated in parentheses. These designations are as follows: type 1B (type I) for SCCmec elements carrying type 1 ccr and class B mec, type 2A (type II) for SCCmec elements carrying type 2 ccr and class A mec, type 3A (type III) for SCCmec elements carrying type 3 ccr and class A mec, type 2B (type IV) for SCCmec elements carrying type 2 ccr and class B mec, and type 5C (type V) for SCCmec elements carrying type 5 ccr and class C mec.

The second feature of our system is a description of J regions, which could be indicated after the SCCmec type designation with numbers representing differences in the J1 region and J2-J3 regions, each separated by a period, e.g., 2A.1.1. The subtypes of type 2A and type 2B SCCmec elements would be described in numerals in the order that they are found, as follows (previous names are indicated in parentheses): type 2A.1 (type IIa), type 2A.2 (type IIb), type 2B.1 (type IVa), type 2B.2 (type IVb), type 2B.3 (type IVc), and type 2B.4 (type IVd). The structures of five SCCmec types and their subtypes are shown in Fig. Fig.1.1. They are indicated with the proposed new names.

The third feature of the system concerns enumeration. The ccr type or J-region identity could be numbered only in chronological order according to the time of identification. The objective of the new nomenclature is to resolve problems in naming of SCCmec elements. In 2001, Oliveira et al. reported the ccrA4 and ccrB4 genes, which shared weak similarity with three previously reported ccr genes found in a pediatric strain, HDE288 carrying class B mec, and designated the new SCCmec element type IV SCCmec (30). Independently, we found SCCmec elements carrying class B mec and type 2 ccr in C-MRSA strains isolated at the University of Chicago Children's Hospital and designated these type IV SCCmec (22). Since MRSA strains carrying the latter elements are found mostly in C-MRSA strains isolated in the United States or Europe, many researchers have regarded the elements carrying type 2 ccr and class B mec as type IV SCCmec. If the proposed nomenclature were adopted, the type IV SCCmec element found in HDE288 would be called type 4B. As an alternative to roman numerals, the element carried by HDE288 would be the prototypic type IV, since it was reported in 2001. However, at present, type IV SCCmec is regarded as an element that carries class B mec and type 2 ccr, and type V SCCmec is regarded as an element carrying class C mec and type 5 ccr. We suggest that it is reasonable to regard the element that is carried by HDE288 as a sixth ccr and mec combination, consisting of type 4 ccr and class B mec.

Similarly, if we are allowed to rename the SCCmec elements reported by other researchers (20, 23, 30, 36), we would like to do so as shown in Table Table2.2. For example, type IA SCCmec, which carries plasmid pUB110 near mecA gene, could be renamed type 1B.1.2 (or type I.1.2). In this name, 1B (or I) signifies the combination of ccr type and mec class, “1” signifies that the J1 region carrying the pls gene is the same as that carried by strain NCTC10442, and “2” signifies that there is a difference concerning the presence of pUB110 in the J2 and J3 regions with respect to that carried by NCTC10442. As one of seven variants, SCCmec type IIB was reported by Shore et al. (36), which could be renamed 2A.3.2 (II.3.2). In this designation, 2A (or II) signifies the combination of ccr type and mec class, “3” signifies that the J1 region is not the same as that carried by previously determined type 2A SCCmec elements, and “2” signifies that this is the second type identified among type 2A.3 elements. Accordingly, the type IIB SCCmec element reported by Shore et al. and the type IIb SCCmec reported by Hisata et al. (12) can be distinguished easily by describing them as 2A.3.2 (II.3.2) and 2A.2 (II.2), respectively.

TABLE 2.
Uniform nomenclature for SCCmec elements

Identification of types of SCCmec elements distributed in Asian MRSA strains.

All 615 strains tested by PCR were mecA positive, and their SCCmec types were assigned by PCR. The ccr types and mec classes were determined by the procedures and primer sets listed in Table Table1.1. As shown in Table Table3,3, 610 of 615 (99.2%) of MRSA strains could be classified with respect to four types of SCCmec elements: type 3A, 370 strains; type 2A, 207; type 2B, 32; type 1B, 1. Among five strains that could not be classified into any of the four types, three carried type 2 ccr, and two did not carry any ccr genes identified so far by PCR with three sets of primers listed in Table Table11 for ccr typing, for identification of ccr genes, and for ccrC.

TABLE 3.
Distribution of SCCmec elements in 11 Asian countries

Type 3A SCCmec strains predominated in isolates from nine countries: Saudi Arabia, 18/19 strains (94.7%); India, 37/37 strains (100%); Sri Lanka, 29/29 strains (100%); Singapore, 87/87 strains (100%); Indonesia, 60/60 strains (100%); Thailand, 49/49 strains (100%); Vietnam, 34/36 strains (94.4%); Philippines, 14/15 strains (93.3%); and China, 40/40 strains (100%). This type was minor in Korean strains, 2/105 strains (2.0%), and it was not identified in Japanese strains. Type 2A SCCmec strains predominated in Japan and Korea, 126/138 strains (91.3%) and 79/105 strains (75.2%), respectively. The frequency of type 2B SCCmec strains was very low (5.3%), and they were identified in four countries: Saudi Arabia (1 strain), Philippines (1 strain), Korea (24 strains), and Japan (6 strains).

Subtyping of the J1 region and the mec left extremity.

The type 2A and type 2B SCCmec elements could be further subtyped by differences in the J1 regions. The majority of type 2A SCCmec strains (189/207 strains [91.3%]) belonged to subtype 1 (formally designated subtype a). One hundred nineteen of 126 (94.4%) Japanese strains and 70 of 79 (88.6%) Korean strains carried the type 2A.1 SCCmec element. A classification of type 2A SCCmec element carried by the other nine Korean strains and seven Japanese strains could not be determined by PCR with primer sets specific for subtypes 1 and 2 (formally designated subtype b). In the case of the Korean strains, 12 of 24 were type 2B.1 SCCmec and 8 of 24 were type 2B.3 SCCmec, while all six Japanese strains were type 2B.3 SCCmec. The remaining six type 2B strains were unclassifiable when tested with the primer sets specific for four subtypes (subtypes 1, 2, 3, and 4) of type 2B SCCmec elements.

We have previously reported that nucleotides at the right extremities of three SCCmec elements (type I, type II, and type III) are characteristic, and we have developed a PCR method to distinguish SCCmec elements with a set of three primers that recognize the chromosomal region that is common to all S. aureus strains and the others recognizing regions specific for each type that are located at the right extremity of SCCmec elements. We had previously designated this method as mec right extremity polymorphism (MREP) typing (14). However, after the determination of the whole-genome sequences of strains N315 and Mu50, we realized that the SCCmec elements are located downstream of orfX on the chromosomal map. To be consistent with published genome maps, we have revised the illustration of the structure of SCCmec elements by placing orfX at the left as shown in Fig. Fig.1.1. Hence, the region just downstream of orfX, which we initially called the mec right extremity, is now regarded as the left extremity of the SCCmec element. Accordingly, MREP is now referred to as mec left extremity polymorphism (MLEP) typing. In the present study, we performed MLEP testing for all 615 MRSA isolates and compared the left extremities to SCCmec types. We found that 203 of 207 (98.0%) of type 2A SCCmec strains possessed MLEP type ii, 320 of 370 (86.4%) type 3A SCCmec strains possessed MLEP type iii, and the sole type 1B SCCmec strain possessed MLEP type ii. These results demonstrate that there is a strong correlation between the SCCmec element type and MLEP type. Among type 2B SCCmec strains, most (28 of 32) carried MLEP type ii, while 3 carried MLEP type i, and 1 is still untypeable.

Among 50 (13.6%) type 3A SCCmec strains that showed discrepancies, 39 (10.5%) carried the type ii left extremity and the other 11 (3.0%) could not be classified as either type. Since this was unexpected, we confirmed this result with a primer set that recognizes the dcs (downstream constant sequence) region or locus D, which has been previously tested by multiplex PCR (31, 32). All 39 strains were confirmed to be positive for the D locus.

A composite of SCCmercury and type 3A SCCmec.

The ccrC gene encodes a site-specific recombinase that is responsible for the mobility of the type 5C (V) SCCmec element by mediating its precise excision and integration of the element into the chromosome (15). However, the localization of the ccrC gene is not restricted in type 5C SCCmec elements. It had been found as CZ072 in the type III SCCmec element of strain 85/2082 (accession no. AB037671) as reported previously (14). Soon after we reported the type III SCCmec element of 85/2082 as being the longest such element (67 kb), we noticed three integration site sequences (ISSs) located in and around the element (Fig. (Fig.2),2), which suggested that the long region could be a composite of two SCC elements, one carrying ccrC and the other carrying ccrA3 ccrB3 as site-specific recombinases (11). In the present study, we carried out PCR to test this idea using validate the presumption using MRSA 85/2082 chromosomal DNA as a template and primers sets designed around ISSs to determine whether the two SCC elements independently excise. No PCR product was obtained in a standard PCR experiment with three primer sets that allow detection of the precise excision of two SCCs (cR1 and cLt4), type 3A SCCmec (mN17 and cLt5), and an SCC carrying ccrC (cR1 and mN19). Therefore, we then tried to identify precise excision by a nested PCR approach and found that DNA fragments containing attB could be amplified, although the frequency of excision of either element alone was lower than that of both elements concomitantly.

These data show that an SCC element carrying the mercury resistance operon and the site-specific recombinase ccrC can be distinguished from type 3A SCCmec elements carrying class A mec and type 3 ccr. Therefore, we have designated this element SCCmercury.

To determine the distribution of SCCmercury, we conducted a PCR experiment to identify the ccrC gene, the mercury resistance determinant, and the junkyard region. The results showed that 371 strains carried the ccrC gene. Among these, type 3A SCCmec was found in 355 strains, of which 313 carried MLEP type iii (Table (Table3).3). Since 310 of 313 strains were positive for PCR to detect the mercury resistance determinant and/or junkyard region, they can be considered to possess an SCCmercury element that is similar to that of strain 85/2082. Other ccrC-positive type 3A SCCmec strains included 34 Chinese strains carrying MLEP type ii, seven Indian strains, and a Singapore strain whose MLEP was untypeable. Interestingly, a Korean type 2A SCCmec strain and 15 Korean type 2B strains carried ccrC. Our PCR analyses have not identified the ccrC gene in the type 2A or type 2B SCCmec element. These strains may carry elements encoding ccrC integrated at a position different from that of orfX (Y. Kondo, unpublished data).

Mercuric chloride was an antiseptic used in the past, and S. aureus originally became resistant to this agent by the acquisition of plasmids encoding mercury resistance determinants, such as pI258 (24, 27, 35). Nowadays, mercuric chloride is no longer widely used in hospitals, and the mechanism of acquisition of SCCmercury by S. aureus has not been clarified. One possibility is that a coagulase-negative staphylococci (C-NS) acquired a mercury resistance determinant that permitted survival under the pressure of mercuric chloride, and this determinant may have integrated into an SCC element to form SCCmercury in C-NS, from which it was transferred to S. aureus. Alternatively, a plasmid-encoded determinant may have been transferred to S. aureus and it then integrated into an SCC element to form SCCmercury. SCCmercury may be a good example of an SCC element that can acquire exogenous genes to promote the survival of staphylococci under selective pressures. It is plausible that MSSA strains carrying an element encoding ccrC or similar to SCCmercury were preexisting and were changed to MRSA strain by the integration of an SCCmec element. Dominant strains carrying both type 3A SCCmec and SCCmercury, as represented by strain 85/2082, may have arisen by the integration of type 3A SCCmec into MSSA strains carrying SCCmercury.

Identification of two small type 3A SCCmec elements.

We speculated that if the type III SCCmec element of strain 85/2082 is a composite of two SCC elements, there may be strains that carry only the type 3A SCCmec element. To identify such strains, we conducted PCR experiments and identified two small type 3A SCCmec elements whose junkyard regions downstream of orfX were different (Fig. (Fig.2).2). One was found by screening 11 MLEP untypeable strains by PCR with the primer set cR2 (specific for orfX) and mN19 (located downstream of DR2). A DNA fragment of 1 kb was successfully amplified only from the Singaporean isolate JCSC290. Subsequently, the entire region of the type 3A SCCmec element in JCSC290 was amplified by long-range PCR with primer sets specific for essential constituents of the type 3A SCCmec elements of 85/2082. The size of the type 3A SCCmec element carried by JCSC290 was almost identical to that of 85/2028 as judged by agarose gel electrophoresis. The nucleotide sequences of both ends of the JCSC290 type 3A SCCmec element were identical to the corresponding ends of the type 3A SCCmec element of 85/2082 (Fig. (Fig.22).

The other element was identified in the Saudi Arabian isolate JCSC1716, for which we had tried to determine the structure of SCCmec element that lacked ccrC but carried MLEP type ii. The entire region of the type 3A SCCmec element carried by JCSC1716 was approximately 31 kb in size, as estimated by long-range PCR. Two direct repeats and incomplete inverted repeats were also identified at the ends of the element (Fig. (Fig.22).

We also carried out PCR to investigate whether the type 3A SCCmec elements precisely excise from the chromosome. When chromosomal DNAs of strains JCSC290 and JCSC1716 were used as templates, a 0.35-kb DNA fragment containing attBscc was successfully amplified, indicating that small type 3A SCCmec elements can be excised.

According to the new nomenclature, the SCCmec element carried by strain JCSC290 is called type 3A.1.1, and the element carried by strain JCSC1716 is called type 3A.1.4. Oliveira et al. reported two type III SCCmec variants, IIIA and IIIB (30). Since type IIIA does not carry plasmid pT181 in the J3 region, it may be regarded as type 3A.1.2. As for type IIIB, it closely resembles the SCCmec element carried by JCSC290 except that it does not carry plasmid pT181. Since this element was reported in 2001, it may be regarded as type 3A.1.3., indicating that it is the third variant found with differences in the J2 and J3 regions of type 3A.1 SCCmec. The IIIA and IIIB types would differ in the presence of ips (the region between the IS431 right flanking copy of pT181 and the IS431 left flanking copy of pI258), if the SCCmercury region is omitted from the illustrated structure of type IIIA SCCmec. Furthermore, it is noteworthy that the type 3A SCCmec elements are not large and that previously reported type III SCCmec strains have a long region composed of two SCCmec elements.

Characteristics and evolution of Asian MRSA clones.

The genotypes of representative isolates were determined by multilocus sequence typing. As shown in Table Table4,4, three strains that carry dominant SCCmec types and as many strains that carry minor SCCmec types in 11 countries were chosen and their genotypes were determined by MLST. MLST analysis revealed that the dominant MRSA strains in Korea and Japan belonged to clonal complex 1 (CC1) or CC5, whereas the dominant MRSA strains of the other nine countries belonged to CC8 (Table (Table4).4). CC5 type 2A SCCmec clones predominate in Korea and Japan, and CC8 type 3A SCCmec clones predominate in the other nine countries as reported by Ko et al. (19).

TABLE 4.
Genotypes of representative MRSA strains assigned by MLST

When we investigated SCCmec types in detail, we noticed that the MRSA clones distributed in Asian countries were surprisingly diverse and that they exhibited characteristic features in each country. We tested whether CC5 type 2A SCCmec strains carry the ant(4′)-1 gene near the mecA gene and found that the frequency of this gene differed among strains isolated from two countries. Among 126 Japanese type 2A.1 strains, 121 (96%) carried the ant(4′)-1 gene downstream of mecA, whereas only 42 of 79 (53%) Korean type 2A.1 SCCmec strains carried it. These data agree well with that of Cha et al., who reported that 32 of 60 (53.3%) type 2A.1 SCCmec strains did not carry plasmid pUB110, and they called this element a type II variant (4). The type II SCCmec element that does not carry plasmid pUB110 can be renamed type 2A.1.2 (II.1.2) according to our proposed nomenclature.

Among the minority clones in each country, most were type 3A SCCmec strains, other than a Singapore isolate belonging to CC8. Other minor clones carrying type 2A or type 2B SCCmec elements belonged to CC1, CC5, CC8, or CC36.

The MLST genotypes of Korean and Japanese MRSA strains carrying SCCmec elements other than type 2A.1 did not always differ from those of major clones, suggesting that these MRSA clones might have been generated by the integration of an SCCmec element that was distributed in each country as a major MSSA clone. When we closely examined CC8 type 3A SCCmec clones, we noticed that each was composed of a dominant clone ST239 carrying a composite of type 3A SCCmec and SCCmercury elements and a minor clone ST239 carrying type 3A SCCmec, whose junkyard region was not always conserved, and different SCC elements represented by strains JCSC1716, JCSC298, JCSC234, JCSC235, JCSC1570, JCSC1675, and the Chinese dominant clone. It is possible that both SCCmercury and type 3A SCCmec elements had produced many variants and that these variants had integrated into MRSA strains or had been disseminated in each country. Strains carrying a type 3A SCCmec element alone, such as JCSC290 and JCSC1716, may have been generated either bythe integration of a type 3A SCCmec element into MSSAstrains prevalent in each country or by the excision of SCCmercury.

As for Korean CC8 strains, a strain belonging to ST344 carried type 3A SCCmec element. These data quite agree well with those of Cha et al., who showed that Korean type III SCCmec strains belonged to ST239 or ST344 (4). In contrast, other strains carrying minor SCCmec elements such as 2A.n, 3A, 2B.1, 2B.3, and 2B.n belonged to ST254 and ST345. Interestingly, most of these type 2A SCCmec and type 2B SCCmec strains belonging to ST254 carried the ccrC gene. The reason why so many different SCCmec elements are integrated into ST254 strains remains unclear. Since ST254 was identified only in Korean strains in this study, we presume that Korean ST254 strains acquired the SCCmec element and were locally changed to MRSA strains.

It has been hypothesized that a certain SCCmec element was integrated into MSSA cells to generate a MRSA clone and that the SCCmec element was excised to generate MSSA, and another SCCmec element was integrated in it to form another MRSA clone (8). This might be one hypothesis for the evolution of MRSA. However, it was reported that methicillin-resistant determinants create large deletions upon excision (34, 38). As far as we can tell, the spontaneous precise excision of the SCCmec element from the MRSA strain is infrequent, and the frequency of the precise excision of mecA from IS431 is approximately 1/10th that of the deletion frequency (Y. Katayama, N. Aritaka, et al. unpublished data). Another possibility is that SCCmec carried by methicillin-resistant C-NS might be integrated into MSSA to generate MRSA clones, but this does not directly conflict with the above hypothesis.

Concerning the distribution of major clones, the data agree very well with the previous reports that type 2A SCCmec strains are dominant in Korea and Japan and that type 3A SCCmec strains are dominant in the other nine countries (4, 8, 14, 19, 30). What is the reason for these geographical differences among MRSA clones? The explanation may lie in the fact that staphylococci inhabit the skin and mucous membranes of healthy humans and may be transported to other countries that are culturally or economically related. The data may suggest that these relations may influence the distribution of staphylococci as well as MRSA strains, since the CC5 type 2A1 SCCmec clone has been found in Korea, Japan, and the United States and the CC8 type 3A SCCmec clone is distributed in Asian countries where the influence of European countries is considerable. A worldwide analysis of MRSA clones and the study of the mechanism of acquisition of SCC elements from other species should help resolve these questions in the near future.

SCCmec typing in the past and in the future.

Oliveira and Lencastre developed a multiplex PCR system to assign four types of SCCmec elements by identifying characteristic genes or motifs in junkyard regions (31). However, the use of the multiplex PCR for assignment of SCCmec elements raises some concerns. First, the presence of locus D (dcs region) is not confined to type 1B (type I), type 2A (type II), and type 2B (type IV) SCCmec elements. We showed in this study that locus D can be identified in 39 strains carrying type 3A SCCmec element. Second, the identification of type III SCCmec is problematic. We have reported that the type III SCCmec element carried by MRSA 85/2082 is the longest SCCmec element. Oliveira and Lencastre might agree with our concept and the element they have used as type III SCCmec turns out to be a composite of type 3A SCCmec and SCCmercury. Therefore, it could be said that identification of two loci that are used to assign type III SCCmec element, locus E (the region between the mercury operon and Tn554) and locus F (ccrC) shown in Fig. Fig.1,1, signifies the carriage of SCCmercury. Third, types of SCCmec elements would not be determined by assigning J regions. There are many type 2A SCCmec elements which do not carry the kdp operon in the junkyard region and these would not be classified as type II by the multiplex PCR system. Another multiplex strategy for SCCmec assignment has been reported by Zhang et al. (40) and further developed by our group (Y. Kondo, T. Ito, X. X. Ma, S. Watanabe, B. N. Kreiswerth, J. Etienne, and K. Hiramatsu, unpublished data). Both systems share the strategy to identify mec, ccr, and the J1 region, although they differ slightly with respect to primer sequences and target genes. We expect that SCCmec elements could be assigned by first determining the mec class and ccr type and then by identifying differences in J regions with the sets of primers reported previously (12, 31, 40) or described in this paper.

With the increasing interest in MRSA, it is likely that numerous SCCmec elements will be found in the near future. Since establishing a new nomenclature requires the consensus of many dedicated staphylococcal researchers, a meeting is needed to solve the problematic issue of naming SCCmec elements. We proposed the usage of roman numerals as an alternative because they are simple, well-known, and already accepted. We have attempted to establish a basis for the nomenclature of SCCmec elements, and we invite the suggestions of the staphylococcal research community for improvements in the proposed nomenclature.

Acknowledgments

We thank Mark C. Enright for his kind help with MLST.

This work was supported by a Grant-in-Aid for 21st Century COE Research and a Grant-in-Aid for Scientific Research on Priority Areas (13226114) from The Ministry of Education, Science, Sports, Culture and Technology of Japan.

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