ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2006, p.

34573459
0066-4804/06/$08.000 doi:10.1128/AAC.00629-06
Copyright 2006, American Society for Microbiology. All Rights Reserved.

Vol. 50, No. 10

Redefining a Structural Variant of Staphylococcal Cassette

Chromosome mec, SCCmec Type VI
Duarte C. Oliveira,1* Catarina Milheirico,1 and Hermnia de Lencastre1,2
Laboratory of Molecular Genetics, Instituto de Tecnologia Qumica e Biolo
gica (ITQB), Universidade Nova de Lisboa,
Oeiras, Portugal,1 and Laboratory of Microbiology, The Rockefeller University, New York, New York 100212
Received 23 May 2006/Returned for modification 2 July 2006/Accepted 16 July 2006

Previously we identified a SCCmec variant similar in size to type IV but with a new ccrAB allotype, allotype
4. We addressed the epidemiological relevance of this variant and found it among several strains belonging to
the same clone. We propose to rename this structural variant SCCmec type VI.
Methicillin-resistant Staphylococcus aureus (MRSA) is characterized by a large heterologous mobile genetic elementthe
staphylococcal chromosomal cassette, or SCCmec (7)carrying the mecA gene, the central element of methicillin resistance, and the ccrAB locus, which encode recombinases involved in SCCmec mobility (11). SCCmec types are defined by
combining the information on the genetic organization of the
mec complex with the ccrAB allotype (6, 15). In S. aureus, three
major mec complex classes have been described: class A contains the complete mec complex (mecI-mecR1-mecA), and
classes B and C have the mecA regulatory genes disrupted due
to the presence of insertion sequences (IS1272-mecR1mecA and IS431-mecR1-mecA, respectively) (8, 10). Concerning the ccrAB locus, three major allotypes (ccrAB1 to
ccrAB3) (6) and one sporadic allotype (ccrAB4) (17) have been
identified. Recently, a new type of ccr gene complex, which
consists of only one gene (ccrC) not closely related to the ccrA
or ccrB gene, was reported (8). SCCmec carries other sequences that define the overall genetic organization of the
resistance cassette. These regions may be used as targets for
typing strategies (1, 16, 22), and polymorphisms within these
regions, particularly in the region downstream of the ccrAB
genes (the J1 region), define SCCmec subtypes or variants (9,
12, 15, 17, 21).
After the description of SCCmec types I, II, and III (6),
almost simultaneously two groups reported on the existence of
a fourth structural type, named SCCmec type IV by both studies (12, 17). However, although those SCCmec elements are
similar in size and share the same class of mec complex and the
same genetic organization in the mecA downstream vicinity,
they differ in the ccrAB allotype (ccrAB2 versus the previously
unidentified ccrAB4) and in the J1 region. Later, SCCmec type
IV with ccrAB allotype 2 was shown to be dominant among the
emerging community-acquired MRSA strains, stressing its epidemiological relevance, whereas ccrAB allotype 4 remained
extremely rare. Recently, ccrAB complexes closely related to
ccrAB4 have been identified in a composite SCC structure in a
Staphylococcus epidermidis strain (14) and in a MRSA clone

belonging to clonal complex 45 and circulating in Zurich, Switzerland (18).

Strain HDE288 is the prototype strain for the SCCmec characterized by ccrAB4, and its SCCmec element has previously
been fully characterized by PCR screenings and nucleotide
sequencing (17). This strain is also the prototype of the socalled pediatric MRSA clone (sequence type 5), which was
dominant in a pediatric hospital in Portugal in 1992 (19). The
pediatric MRSA clone has also been detected in Poland,
Argentina, Colombia, and the United States (4, 13, 19). In
order to access the epidemiological relevance of this SCCmec
element characterized by ccrAB4, we assembled a collection of
MRSA strains belonging to the pediatric clone from several
hospitals and cities in Portugal and also from international
sources (Table 1).
All strains were initially typed by the SCCmec multiplex
PCR strategy (16) and presumptively assigned to SCCmec type
IV: class B for mec complex (mecI negative), mecA downstream vicinity typical of SCCmec types I, II, or IV (dcs positive), and mecA upstream vicinity negative for elements specific for SCCmec types I to III. Assignment to the class B mec
complex (IS1272-mecR1-mecA) was confirmed for all
strains by PCR with the following primers specific for IS1272
and mecR1 (the location relative to the sequence of
NCTC10442 [6], GenBank/EMBL/DDBJ accession number
AB033763, is given in parentheses after the primer sequence):
ISF1 (AAT TGA AGC AAA TGC CAA TCG) (positions
28812 to 28832) and MRP1 (CAA CTG TCG TAG TCG AAA
CC) (positions 30734 to 30715). All strains were positive, with
an amplicon similar in size to that of the prototype strain of
SCCmec type I, characterized by a class B mec complex (6).
ccrAB typing was then performed by PCR detection with primers specific for ccrAB allotypes 2 (15) and 4 (ccrAB4 F1 [TCA
TCA ATA AGT ATG GAA CG] and ccrAB4 R1 [TTT CTT
GCG ACT CTC TTG G]). Strains with ccrAB2 were classified
as SCCmec type IV.
Strains positive for ccrAB allotype 4 were further characterized for the mecA upstream vicinity with primers designed
based on the previously determined sequence of strain HDE288
(GenBank/EMBL/DDBJ accession number AF411935) (17). The
primer sequences are as follows (with the location relative to the
HDE288 sequence given in parentheses where appropriate):
HDP9, CCC TCC AAA TTA TTA TCT CC (positions 54 to 73);

* Corresponding author. Mailing address: Laboratory of Molecular

Genetics, Instituto de Tecnologia Qumica e Biolo
gica (ITQB), Rua
da Quinta Grande, 6, 2780-156 Oeiras, Portugal. Phone: 351 21 446
9862. Fax: 351 21 442 8766. E-mail: dco@itqb.unl.pt.
3457

3458

NOTES

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1. Relevant characteristics of the pediatric MRSA strains included in this study a
spa typing result

Strainb
code

Originc

Yr(s) of
isolation

mec
complex

ccrAB
allotype

SCCmec
type

HDE1
HDE5
HDE65
HDE71
HDE91
HDE163
HDE174
HDE232
HDE255
HDE266
HDE287
HDE288
HDE362
HDE373
HDE383
HPV17
HPV99
IPO92
HUC136
VNG17
FFP311
RJP17
HSA74
COB5
CLB1
COB3
COB35
COB43
COB62
COB96
COB109
ARG9
ARG299
ARG164
BM18
BM26

Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Lisbon, Pt.
Coimbra, Pt.
Vila Nova de Gaia, Pt.
Oporto, Pt.
Oporto, Pt.
Oporto, Pt.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Bogota, Cob.
Buenos Aires, Arg.
La Plata, Arg.
Buenos Aires, Arg.
New York, NY
New York, NY

1992
1992
19931994
19931994
19931994
1995
1995
1995
1995
1995
1996
1996
1996
1997
1997
19921993
19921993
2001
1995
19921993
1996
19921993
1993
1997
1996
1996
1997
1997
1997
1998
1998
1996
1996
19941996
1989
1989

B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
VI
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV

Egenomicse
type

Ridom f
type

TJMBDMGMK

45

t311

TJMBDMGMK

45

t311

TJMBDMGMK

45

t311

TJMBDMGMK

45

t311

TJMBDMGMK

45

t311

TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBDMGMK
TJMBMDMGMK

45
45
45
45
45
45
45
45
2

t311
t311
t311
t311
t311
t311
t311
t311
t002

TJMBMDMGMK
TMDMGMK

2
47

t002
t045

TJMBMDMGMK
TJMBMDMGMK
TJMBMDMGMK

2
2
2

t002
t002
t002

Sequence
repeat

Sequence type
(MLST)

5
5
5
5

All strains have been previously assigned to the pediatric MRSA clone based on PFGE patterns, ClaI::mecA patterns, and ClaI::Tn554 insertion patterns (19).
Strain letter codes reflect strain origins (hospital or country) as follows: HDE, Hospital Dona Estefania, Lisbon, Portugal; HPV, Hospital Pulido Valente, Lisbon,
Portugal; IPO, Instituto Portugues de Oncologia, Lisbon, Portugal; HUC, Hospital da Universidade de Coimbra, Coimbra, Portugal; VNG, Hospital de Vila Nova de
Gaia, Vila Nova de Gaia, Portugal; FFP, Faculdade de Farmacia do Porto, Oporto, Portugal; RJP, Instituto Dr. Ricardo Jorge, Oporto, Portugal; HSA, Hospital de
Santo Anto
nio, Oporto, Portugal; COB or CLB, Colombia; ARG, Argentina; BM, New York Hospital Medical Center of Queens, New York, NY.
c
Country names are abbreviated as follows: Pt., Portugal, Cob., Colombia; Arg., Argentina.
d
spa sequence repeats are given according to the nomenclature proposed by B. N. Kreiswirth and colleagues (20).
e
spa types assigned by Egenomics software (B. Kreiswirth and S. Naidich, personal communication).
f
spa types assigned by Ridom software (5).
b

HDP17, GCA ATT AGT TAC AAA GCA GC (positions 2833

to 2814); HDP18, CAT CTT CAA AGA CTT TTA GTC C
(positions 2461 to 2482); HDP8, ACT AAC GGT AAA ACA
TGA CC (positions 6925 to 6904); HDP2, TTA AAA GAT
GCC AAC GAA GG (positions 9382 to 9401); ISR2, ATT
CGT CGA ATT CAT TGT CAG G (specific for IS1272
outwards). Primers HDP9 and HDP17 amplify within the J1
region close to the chromosomal junction; primers HDP18 and
HDP8 amplify within the J1 region close to the ccrAB locus;
and primers HDP2 and ISR2 amplify the region between the
ccrAB locus and IS1272. PCRs were performed in a T1 thermocycler (Biometra, Goettingen, Germany) under the following conditions: 94C for 4 min; 30 cycles of 94C for 30 s, 55C
for 30 s, and 72C for 30 s or 2 min; and a final extension at
72C for 4 min. In each reaction (final volume, 50 l), 5 ng of
chromosomal template, 1.25 U of Amplitaq DNA polymerase

(Applied Biosystems, Foster City, CA), and 20 pmol of each

primer were used in 1 PCR buffer with MgCl2 at 1.5 mM
(Applied Biosystems) and a deoxynucleoside triphosphate mix
at 0.16 mM (MBI Fermentas, Hanover, MD). All ccrAB4
strains gave positive signals with the expected size after PCR
amplifications with the three primer pairs.
The SCCmec type characteristic of strain HDE288 was
found in all 15 isolates from the same hospital (Hospital Dona
Estefania, Lisbon, Portugal) and in 4 other isolates from three
hospitals, two located in the same city (Hospital Pulido
Valente and Instituto Portugues de Oncologia, Lisbon, Portugal) and one located in a different city (Hospital da Universidade de Coimbra, Coimbra, Portugal). The remaining
Portuguese isolates and all international isolates were characterized by ccrAB allotype 2. Since the initial assignment of
strains to the pediatric MRSA clone was based on pulsed-field

VOL. 50, 2006

NOTES

gel electrophoresis (PFGE) patterns, we have further characterized the genetic background of some strains by spa typing
and multilocus sequence typing (MLST), as previously described (2, 3, 5, 20). All strains analyzed were characterized by
sequence type 5 and by closely related spa types (TJMBD
MGMK motif, type 311) (Table 1).
These results show that the SCCmec type previously found
only in strain HDE288 is also present in other local strains
belonging to the same clone, as defined by PFGE and confirmed by spa typing and MLST. Interestingly, most strains
belonging to the pediatric MRSA clone are not characterized
by this SCCmec type, suggesting at least two acquisitions of the
mecA gene in the same genetic background. Still, the SCCmec
type found in strain HDE288, defined by a class B mec complex, ccrAB allotype 4, and a specific J1 region, is epidemiologically relevant, and therefore we propose that it be identified as SCCmec type VI or type 4B, according to new SCCmec
nomenclature proposed by Chongtrakool et al. (1).
Partial support for this study was provided by project POCI/BIAMIC/58416/2004 from Fundacao para a Ciencia e Tecnologia (FCT),
Lisbon, Portugal, and by project 55068 from Fundacao Calouste
Gulbenkian, Lisbon, Portugal, both awarded to H.D.L. D.C.O. and
C.M. were supported by grants SFRH/BPD/9374/2002 and SFRH/BD/
23010/2005, respectively, from FCT, Lisbon, Portugal.
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