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Biochemical and Biophysical Research Communications 377 (2008) 752756

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Biochemical and Biophysical Research Communications

j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c

Structure and specific detection of staphylococcal cassette chromosome mec

type VII
Wataru Higuchi a, Tomomi Takano a, Lee-Jene Teng b, Tatsuo Yamamoto a,*

Division of bacteriology, Department of Infectious Disease Control and International Medicine, Niigata University Graduate School of Medical and Dental Sciences, 1-757,
Asahimachidori, Niigata 951-8510, Japan
National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan

a r t i c l e

i n f o

Article history:
Received 2 October 2008
Available online 14 October 2008

Methicillin-resistant Staphylococcus aureus
Staphylococcal cassette chromosome mec
type VII (SCCmecVII)
The cassette chromosome recombinase C
gene (ccrC)

a b s t r a c t
Staphylococcal cassette chromosome mec (SCCmec) type VII, found in community-acquired methicillin-resis
tant Staphylococcus aureus belonging to multilocus sequence type (ST) 59 from Taiwan, was 41,347bp in size
and flanked by 19-bp attL and attR sequences. It was inserted into the att site at the 39-end of orfX in the orfXorfY (putative tRNA dihydrouridine synthase) region in ST59 S. aureus. The 59-end side 9911-bp core region
of SCCmecVII, which contained attL and the cassette chromosome recombinase gene (ccrC8), was shared by
other SCC structures, SCCmercury and mosaic SCCmec from Switzerland, indicating its important role in SCC
evolution. The central 21,245-bp core region contained mec complex (C2b) and another ccrC gene (ccrC2),
and was highly homolog
ous to SCCmecV, but with substitutions, insertion and replacement. The 39-end side
10,191-bp sequence was unique. Therefore, SCCmecVII has emerged through recombination and insertion
events. Multiplex and real-time PCR assays were developed for specific detection of SCCmecVII.
2008 Elsevier Inc. All rights reserved.

Staphylococcal cassette chromosome mec (SCCmec) is a mobile

genetic element, which confers on its host resistance to b-lactam
antimicrobial agents, such as penicillins and cephems [1,2]. It con
sists of mec complex (a region containing mecA, mecR, IS/mecI, IS431),
ccr complex (a region containing recombinase genes such as ccrA,
ccrB, or ccrC), and directly repeated 15-base core sequences at both
ends, and is inserted into the attachment site (att) located at the very
end of an open reading frame (ORF), orfX of unknown function, in
methicillin-susceptible Staphylococcus aureus (MSSA) [13]. Seven
types of SCCmec (SCCmecI to SCCmecVII) have been described based
on differences in structure (mec complex and ccr complex) and size
for methicillin-resistant S. aureus (MRSA) [1,38].
Of those, e.g., SCCmecII or SCCmecIII are mainly associated with
hospital-acquired MRSA (HA-MRSA) [1,9]. It has been considered
that HA-MRSA emerged from MSSA by acquisition of SCCmec only
several times, and a relatively small number of pandemic MRSA
clones spread worldwide, such as New York/Japan clone (multilo
cus sequence type [ST] 5, SCCmecII) and Hungarian clone (ST239 and
SCCmecIII) [10]. In contrast, SCCmecIV or SCCmecV are mainly asso
ciated with community-acquired MRSA (CA-MRSA) [5,9]. CA-MRSA
often produce Panton-Valentine leucocidin (PVL); examples are
predominant clones in the United States (genotype: ST8/SCCmecIV
[USA300]) or in Europe (genotype: ST80/SCCmecIV) [9,11,12].
For PVL-positive ST59 CA-MRSA in Taiwan, the SCCmec was
initially classified as SCCmecVT, based on the genetic information
* Corresponding author. Fax: +81 25 227 0762.
E-mail address: (T. Yamamoto).
0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved.

(mec complex C2 and ccr complex C2) obtained from partial

sequence data [13]. Then, it was assigned as a novel SCCmec type
VII, based on the finding (unique feature of the presence of two
ccrC genes [ccrC2 and ccrC8]) obtained from more precise sequence
data [8]. In this study, the entire structure of SCCmecVII was deter
mined and its evolutional characteristics were investigated. Based
on the unique structure, a specific detection method of SCCmecVII
was developed.
Materials and methods
Bacterial strains. PVL-positive ST59 CA-MRSA (23 strains) from
Taiwan, including strains PM1 (from which, partial SCCmecVII
sequence was obtained [8]), TSGH17 (for which, SCCmecVT was
proposed [13]), SSF17, and Ce7, were described previously [8].
Strain TSGH17 and strains SSF17 and Ce7 were kindly provided by
C.C. Wang and M. Ling and L.K. Siu, respectively. PVL-positive ST59
CA-MRSA strains (10 strains), isolated in 2006 from Taiwan, were
also employed. Standard strains used for SCCmec typing included
strains 10442 (for SCCmecI), N315 (for SCCmecII), 85/2082 (for
SCCmecIII), JCSC1968 (for SCCmecIVa), JCSC1978 (for SCCmecIVb),
JCSC4788 (for SCCmecIVc), JCSC4469 (for SCCmecIVd), and WIS (for
SCCmecV); they were kindly provided by T. Ito and K. Hiramatsu.
Sequencing of SCCmecVII region. Sequencing of the SCCmecVII
region of strain PM1 was performed between two ORFs flanking
SCCmec [2]: orfX and orf (for putative tRNA dihydrouridine synthase)
on the non-orfX side (the latter orf was designated as orfY in this
study). ORF analysis of determined sequences was performed using

W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756


Table 1
List of the primers and probes used in this study.
Target gene or sequence

Primer or probe

PCR product size (bp)

GenBank Accession No. or reference



AB353125, [8]




AB462393, this study





AB462393, this study





AB462393, this study

(2) Primers and probes for real-time PCR assay

mec complex C2(C2a, C2b)



AB462393, this study







(1) Primers for PCR assay


mecC2a-P (FAM)
mecC2b-P (VIC)

the software in silico MolecularCloning (R) GE version 1.5.2 (in silico

biology, Kanagawa, Japan). Homology analysis was performed using
the software BLAST (
Phylogenetic analysis of the ccr sequences. The ccr sequences
were taken from GenBank Accession Nos. AB121219 for ccrC1;
AY894416 for ccrC2; AB037671 for ccrC3; U10927 for ccrC4;
AP006716 for ccrC5; EF190467 for ccrC6; EF190468 for ccrC7;
AB462393 or AB353125 for ccrC8; and AM292304 for ccrC9 (ccrC
of SCCmecZH47). The phylogenetic diversity was analyzed by CLU
STALW (, and the graph
was constructed by using Tree View X.
Cloning of the ccrC2 and ccrC8 sequences. For cloning of the ccrC2
and ccrC8 gene sequences, the ccrC2 and ccrC8 gene sequences
were amplified by PCR, and the PCR products were inserted into
a vector pUC18 (Takara Bio, Shiga, Japan) using In-Fusion system
(Takara Bio); resultant recombinant plasmids were designated
pUC18-ccrC2 and pUC18-ccrC8, respectively.
Multiplex and real-time PCR assay. Primers for PCR (and multiplex
PCR), and primers and probes for real-time PCR, designed in this study,
are listed in Table 1. Electrophoresis was performed in 1.5% agarose
with the molecular size standards (stable 100bp DNA ladder; Sigma,
Tokyo, Japan). For real-time PCR, the probes for the mec complex C2
of SCCmecV (designated as C2a in this study) and the mec complex
C2 of SCCmecVII (designated as C2b in this study), respectively, con
tained the reporter dye FAM (6-carboxyfluorescein; Applied Biosys
tems, Foster City, CA) and VIC at the 59-end. FAM and VIC were
detected at 492516 and 535555nm in wave length, respectively.
The 39-end of the probes contained the quencher dye, a minor groove
binder (MGB) molecule, and non-fluorescent quencher (NFQ) (MGBNFQ, Applied Biosystems). Bacterial DNA was prepared using zirco
nia beads as previously described [14]. Real-time PCR was performed
with an ABI 7900HT sequence detector (Applied Biosystems), follow
ing the manufacturers instructions. The cycling conditions were an
initial single cycle for 2min at 50C, followed by a single cycle for
10min at 95C, and 50 cycles of two-temperature cycling consisting
of 15s at 95C and 1min at 60C.
Entire structure of SCCmecVII
The entire nucleotide sequence of the orfX-orfY (putative tRNA
dihydrouridine synthese) region carrying SCCmecVII of PVL+

AB462393, this study

ST59 CA-MRSA strain PM1 was determined (Fig. 1). The orfX-orfY
region of strain PM1 was 49,137bp in size (GenBank Accession
No. AB462393). SCCmecVII was 41,347bp and flanked by 19-bp
directly repeated sequences (attL and attR). When compared with
ST59 MSSA strain 15585, attR of SCCmecVII was identical to the att
site sequence of strain 15585, while attL of SCCmecVII differed by
two nucleotides (Fig. 1A). orfX and the orfY side region (from attR
to orfY) of strain PM1 were highly (99.2% and 99.9%, respectively)
homologous to orfX and the att-orfY region of strain 15585.
SCCmecVII contained 39 ORFs, and was composed of three
distinct regions (Fig. 1B). The left side ccrC region, which was defined
as a region from attL to three small ORFs (tsORF) next to IS431,
was 9911bp in size, and contained 10 ORFs including ccrC8. Inter
estingly, this region was shared by SCCmercury containing ccrC3;
they were 97.5% homologous (Fig. 1B). The central mec complex
C2-ccrC2 region (21,245bp in size) was homolog
ous to SCCmecV
(Fig. 1B). However, compared with SCCmecV, there existed, e.g., (i)
nucleotide substitutions in IS431 (resulting in truncated transpos
ase in WIS431) in mec complex C2 (mec complex C2 of SCCmecV
and VII was named C2a and C2b, respectively); (ii) insertion of the
1270-bp ISSau4-like sequence (which showed 97.0% homologous
to ISSau4 [1261bp]) into orf21 (such ISSau4-like sequence was also
present in SCCmecII and IVg); (iii) non-synonymous substitutions
in ccrC (resulting in conversion of ccrC1 into ccrC2); and (iv) two
small replacements including replacement in orf33. The 10,191-bp
right side region showed no homology to S. aureus sequences, and
showed a relatively low GC content (Fig. 1C).
Specific detection of SCCmecVII
The characteristic feature of SCCmecVII is the presence of two
ccrC (ccrC2 and ccrC8), WIS431 with truncated transposase in mec
complex C2b, and unique sequence at the right side region (e.g.,
orf35). Specific detection was performed by PCR and real-time PCR,
targeting those features (Fig. 2 and Table 1).
Since there were no MRSA strains which possess ccrC2 alone
or ccrC8 alone, each of the ccrC2 and ccrC8 sequence was cloned
into pUC18. PCR primer sets (ccrC2-F2 and ccrC2-R2) and (ccrC8-F
and ccrC8-R), respectively, specifically detected the ccrC2 and
ccrC8 sequences (Fig. 2A), and produced positive results only for
SCCmecVII+ MRSA strains (Fig. 2A and B). Primer sets (mecC2-F and
mecC2-R) detected mec complex C2 (both C2a and C2b), which was
unique to SCCmecV and VII (and possessed DmecR1 and IS431 or


W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756

Fig. 1. The entire sequence of SCCmecVII, its insertion site in ST59 MSSA, and structural comparison with SCCmercury. In (A), the data of ST59 MSSA strain 15585 were taken
from [2]; GenBank Accession No. EU272082. Homologous regions are shown in shadow. Dots above the attL sequence show divergent nucleotide. In (B), the data of SCC
mercurySCCmecIII and SCCmecV were from [15]; GenBank Accession Nos. AB037671 and AB121219, respectively. Homolog
ous regions between SCCmecVII and SCCmercury
or between SCCmecVII and SCCmecV are shown in shadow, and numbers in shadow show percent homology between the corresponding ORFs. ISSau4 was from IS FINDER
( In (C), GC contents in three regions of SCCmecVII (shown in (B)) are shown.

WIS431; Supplementary Fig. 1), as shown in Fig. 2B. Multiplex PCR

with three primer sets, targeting the ccrC2, ccrC8, and mec complex
C2 (both C2a and C2b), gave all three bands for SCCmecVII+ MRSA,
only one band (for mec complex C2) for SCCmecV+ MRSA, and no
bands for other SCCmecI+ to SCCmecIV+ MRSA (Fig. 2B). PCR primer
sets (orf35-F and orf35-R) and (orf33-F and orf33-R), targeting
the orf35 and orf33 sequences (Fig. 1B), also specifically detected
SCCmecVII+ MRSA (data not shown).
To distinguish between mec complex C2a (of SCCmecV) and
mec complex C2b (of SCCmecVII), real-time PCR was designed
(Supplementary Fig. 1 and Table 1). In this real-time PCR, primer
sets (mecC2-F and mecC2-R) reacted with both C2a and C2b,
and probe mec2Ca-P specifically detected the IS431 sequence of
SCCmecV, while probe mec2Cb-P targeted two single nucleotide
substitutions causing truncated transposase in WIS431, thus spe
cifically detected SCCmecVII (Supplementary Fig. 2).

PVL-positive ST59 MRSA strains (33 strains including strains

TSGH17, SSF17, and Ce7) isolated from Taiwan, were examined by
PCR or real-time PCR, described above. SCCmec of those strains
was previously considered to be SCCmecVT, but it was unambigu
ously shown to be SCCmecVII in any case.
Distribution of the left side ccrC region (attL-ccrC-tsORF) as a core
among SCC structures
The attL-ccrC-tsORF region was shared by SCCmecVII and
CCmercury (Fig. 1B). This attL-ccrC-tsORF region was also found
in SCCmecZH47 isolated in MRSA from Switzerland (Fig. 3), indi
cating its important role as a core in SCC evolution. The ccrC
sequence of SCCmecZH47 was divergent from other ccrC sequences
(Supplementary Fig. 3); it was tentatively named ccrC9. When
three attL-ccrC-tsORF regions of SCCmecVII, SCCmercury, and







SCCmecII+ MRSA (N315)




SCCmecI+ MRSA (10442)

PCR for






PCR for


SCCmecIII+ MRSA (85/2082)

W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756



ccrC8 (562)
mec complex C2 (344)
ccrC2 (257)

Fig. 2. Detection by PCR and multiplex PCR of SCCmecVII. In (A), PCR targeting the ccrC2 and ccrC8 sequences was performed. In (B), multiplex PCR targeting three sequences
(ccrC2, ccrC8, and mec complex C2) was performed.

SCCmecZH47 were compared, there existed IS (IS431) or transposon

(Tn554) next to tsORF (Fig. 2B and 3A), suggesting a possibility that
a region next to tsORF provides sites for recombination. SCCmecVII
and SCCmecZH47 also shared the IS431-mecA region next to the
attL-ccrC-tsORF region, indicating that the 15,350-bp left side attLccrC-IS431-mecA region may also play a role as a core in SCCmec
Moreover, in the 9.9kb left side ccrC regions (attL-ccrC-tsORF),
the 6.5kb region, consisting of ccrC with tsORF on the right side
and three ORFs on the left side, was conserved among several
SCCmec and SCC structures containing ccrC (Supplementary Fig. 4).
Such a conserved ccrC region (6.5kb in size) was designated the
ccrC-carrying unit.
The size of SCCmecVII (41.3kb) was relatively large compared
with SCCmecI (34.3kb; GenBank Accession No. AB033763),
SCCmecII (53.0kb; GenBank Accession No. D86934), SCCmecIII
(35.2kb; GenBank Accession No. AB037671), SCCmecIV (24.2kb;
GenBank Accession no. AB063172), and SCCmecV (27.6kb;
GenBank Accession No. AB121219). The size of SCCmecVI has not
been described [7]. Initially described SCCmecIII was large in size
(66.9kb; GenBank Accession No. AB037671), but it was actually
composed with SCCmercury (31.8kb; GenBank Accession No.
AB037671) and SCCmecIII [15]. The mosaic SCCmec (SCCmecZH47),

mainly consisted of SCCmercury and SCCmecIVc, was 33.7kb in

size [16]. It has been considered that SCCmec of CA-MRSA with
smaller sizes, such as SCCmecIV and SCCmecV, may move more
frequently, compared with SCCmec of HA-MRSA with larger sizes,
such as SCCmecII and previous SCCmecIII (SCCmercurySCCmecIII),
and therefore CA-MRSA has been emerging recently [9,18]. Since
SCCmecVII with a large size is associated with major PVL-positive
ST59 CA-MRSA in Taiwan (previous SCCmecVT [13] was found to
be actually SCCmecVII in this study), this hypothes is remains to be
SCCmecVII was inserted into the att site at the 39-end of orfX in
ST59 MSSA to generate ST59 MRSA. Noto et al. [2] proposed that
the 15-bp core att sequence in MSSA plays an important role in
SCCmec insertion, and the inserted SCCmec has the 15-bp directly
repeated core att sequence at both ends. Indeed, for SCCmecVII, att
was assigned to be 19bp, including this core att sequence, although
attR of SCCmecVII was identical to the expected att site sequence
in ST59 MSSA, while attL was different from the expected att site
sequence by two nucleotides (two single base substitution). In
closed agreement with the previous finding by Noto et al. [2], there
was ORF (for putative tRNA dihydrouridine synthese) (designated
orfY in this study) 7.8-kb away from the att site in ST59 CA-MRSA
strain PM1.
SCCmecVII was composed of three distinct regions. The left side
core region of SCCmecVII, which contained attL, ccrC8, and tsORF,
was shared by other SCC structures, SCCmercury and mosaic

Fig. 3. Structural comparison of SCCmecVII with the mosaic SCCmec (SCCmecZH47). The data of SCCmecZH47 were taken from [16]; GenBank Accession No. AM292304. The data
of SCCmecIVc were from [17]; GenBank Accession No. AB245470. Homologous regions between SCCmecZH47 and SCCmecVII or between SCCmecZH47 and SCCmecIVc are shown
in shadow, and numbers in shadow show percent homology between the corresponding ORFs.


W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756

SCCmec from Switzerland, indicating its important role in SCC

evolution. Next to tsORF, there existed IS (IS431) or transposon
(Tn554). For recombination events of the left side attL-ccrC core
region, tsORF region and IS or Tn may play a role. Moreover, the
ccrC-carrying unit, a smaller structure within the left side attLccrC core region, could also be a core in evolution. The central mec
complex (C2b)-ccrC2 core region of SCCmecVII was homologous to
SCCmecV, and the right end side of SCCmecVII was a unique region.
Therefore, SCCmecVII has emerged obviously through multiple
recombination events.
This study was supported by a grant from the Japan Science and
Technology Agency, Japan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bbrc.2008.10.009.
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