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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
b
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
Keywords:
Methicillin-resistant Staphylococcus aureus
(MRSA)
Staphylococcal cassette chromosome mec
type VII (SCCmecVII)
The cassette chromosome recombinase C
gene (ccrC)
Evolution
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.
W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756
753
Table 1
List of the primers and probes used in this study.
Target gene or sequence
Primer or probe
59-ATAAGTTAAAAGCACGACTCA
59-TTCAATCCTATTTTTCTTTGTG
257
AB353125, [8]
ccrC8-F
ccrC8-R
59-GCATGGGTACTCAATCCA
59-GGTTGTAATGGCTTTGAGG
562
orf33
orf33-F
orf33-R
59-CAAAATCAACGTGTGGGAAA
59-GACTTCTTCCTCTATAGCTTGT
534
orf35
orf35-F
orf35-R
59-CAGAGGCTCATCTACATCCT
59-TGTTCTGCTATACCTTCCACA
304
59-ATCAGTTCATTGCTCACGATATGTGTA
59-CAATACGCCATTTGTAATAAGCTTTT
344
C2a
C2b
59-CTACCGTTGGGTTCAAG
59-CTAGCGTTGAGTTCAAG
Name
Sequence
ccrC2-F2
ccrC2-R2
ccrC8
mecC2a-P (FAM)
mecC2b-P (VIC)
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
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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
(http://www-is.biotoul.fr/IS.html). In (C), GC contents in three regions of SCCmecVII (shown in (B)) are shown.
562
257
755
Marker
Strain
Marker
(bp)
PCR for
ccrC8
PM1
PM1
pUC18-ccrC8
pUC18-ccrC2
Strain
PCR for
ccrC2
pUC18-ccrC8
W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756
pUC18-ccrC2
(bp)
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.
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.
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W. Higuc hi et al. / Biochemical and Biophysical Research Communications 377 (2008) 752756