Journal of Antimicrobial Chemotherapy (2009) 63, 32 41

doi:10.1093/jac/dkn435
Advance Access publication 11 November 2008

Genetic diversity of methicillin-resistant Staphylococcus aureus

carrying type IV SCCmec in Orebro County and the western
region of Sweden
Carolina Berglund1,2*, Teruyo Ito2,3, Xiao Xue Ma3,4, Megumi Ikeda2,3, Shinya Watanabe2,5,
Bo Soderquist1,6 and Keiichi Hiramatsu2,3
rebro University Hospital, O
rebro, Sweden; 2Department of Infection
Department of Clinical Microbiology, O
Control Science, Juntendo University, Postgraduate School, Tokyo, Japan; 3Department of Bacteriology,
Juntendo University, School of Medicine, Tokyo, Japan; 4Department of Medical Microbiology and Parasitology,
China Medical University, Shenyang, China; 5Tuberculosis Research Section, Laboratory of Clinical Infectious
Disease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, USA;
6
rebro University Hospital, O
rebro, Sweden
Department of Infectious Diseases, O
1

Background: Recent studies have shown a predominance of type IV SCCmec among the methicillin rebro County and the
resistant Staphylococcus aureus (MRSA) isolated in the low endemic areas of O
western region of Sweden. However, many of these isolates were not possible to classify as existing
subtypes IVa, IVb, IVc or IVd.
Methods: We analysed 16 such MRSA isolates by multilocus sequence typing, spa typing, staphylocoagulase (SC) typing and detection of type IVg and IVh SCCmec. MRSA that remained as unknown
type IV SCCmec were investigated by long-range PCR covering the J1 region; however, only two isolates were possible to amplify by PCR. The nucleotide sequences of the entire SCCmec of these two
MRSA were determined. In addition, isolates that had unknown SC types were investigated by nucleotide sequencing of the coa genes.
Results: Five of 16 isolates were classified as type IVg SCCmec, and four isolates had type IVh
SCCmec. Two subtypes of type IV SCCmec shared J1 regions previously identified in other types of
SCCmec, types I.2 and II.2. The novel elements were designated as type IVi and IVj SCCmec. In
addition, the genetic backgrounds of these Swedish MRSA were diverse and constituted at least nine
sequence types and eight SC types, including four new types of SC.
Conclusions: Type IV SCCmec is occurring in heterogeneous clones of MRSA in Sweden, and the
majority of the type IV SCCmec were identified in community-acquired MRSA. We describe two novel
subtypes of type IV SCCmec with common J1 regions shared by other types of SCCmec, which indicate that J1 regions occurred as primordial SCC.
Keywords: MRSA, community-acquired MRSA, staphylococcal cassette chromosome mec, staphylocoagulase

antibiotics. SCCmec is integrated at the 30 end of the open

reading frame X (orfX) at the specific site attBSCC located close
to the origin of replication in the staphylococcal chromosome.
SCCmec contains a characteristic combination of two essential
genetic components; the mec gene complex with mecA and its
regulator genes, and the cassette chromosome recombinase (ccr)

Introduction
The staphylococcal cassette chromosome mec (SCCmec) is the
genetic element that carries the methicillin resistance determinant, mecA, that makes it possible for staphylococci to continue cell wall assembly and growth in the presence of b-lactam

.....................................................................................................................................................................................................................................................................................................................................................................................................................................

*Corresponding author. Present address: Department of Microbiology, Aleris MEDILAB, SE 183 15 Taby, Sweden.
E-mail: carolina.berglund@aleris.se
.....................................................................................................................................................................................................................................................................................................................................................................................................................................

32
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Received 13 June 2008; returned 7 August 2008; revised 30 August 2008; accepted 22 September 2008

MRSA with type IV SCCmec in Sweden

rebro County and
Seven of the Swedish MRSA were isolated in O
rebro University Hospital, O
rebro, Sweden, and
were stored at O
nine originated from the western region of Sweden, Sahlgrenska
University Hospital, Gothenburg.

gene complex that provides mobility. The SCCmec allotypes are

classified as I, II, III, IV, V or VI, depending on the combination
of mec gene complex (class A, B or C) and ccr gene complex
(type 1, 2, 3, 4 or 5) contained.1 3 Subtyping of SCCmec is
based on nucleotide differences in the three non-essential junkyard (J) regions.1 The J1 region located between the ccr genes
and the downstream chromosomal region is the most important
region for further subtyping of type IV SCCmec. There are five
major subtypes, IVa (IV.1), IVb (IV.2), IVc (IV.3), IVd (IV.4)
and IVg (IV.5), of the type IV SCCmec, described so far.4 8
A sixth subtype was recently described in EMRSA-15 and was
designated as type IVh (IV.6).9
Characterization of methicillin-resistant Staphylococcus aureus
rebro County, Swedenhas
(MRSA) in a low endemic areaO
revealed a significant diversity and the presence of 19 sequence
types (STs) among a collection consisting of 57 isolates, indicating
that the MRSA have arisen on several different occasions by independent acquisition of the SCCmec.10 In addition, the majority of
these MRSA were isolated from individuals who had not recently
been in contact with the healthcare system and were therefore
classified as community-acquired MRSA (CA-MRSA).10 Similar
findings have been made when investigating a collection of
MRSA from the western region of Sweden.11 The most common
rebro County were ST45, ST80 and ST5, but also
genotypes in O
representatives from the five major pandemic clones have been
sporadically identified.10 MRSA genotypes related to those found
rebro County have also been described in, for example,
in O
Ireland and Denmark, an indication that common MRSA clones
are circulating in northern Europe.12,13
Characterization of CA-MRSA from different countries
shows that they usually carry type IV SCCmec, a small element
that generally does not contain any additional resistance
genes.4,14 SCCmec type IV is also found in various genetic
backgrounds, a finding that suggests that this element is more
mobile than the other types of SCCmec.15 Recent studies have
shown a predominance of type IV SCCmec among the MRSA
rebro and in the western region of Sweden;
isolated both in O
however, many of these isolates are not possible to classify as
subtype IVa, IVb, IVc or IVd.11,16
The aim of this study was to characterize type IV SCCmec that
carried subtypes other than IVa, IVb, IVc or IVd, from MRSA
rebro County and the western region of Sweden by
isolated in O
molecular methods and nucleotide sequencing of the SCCmec.

Susceptibility testing
The MICs of different antimicrobial agents were determined by the
agar dilution method according to CLSI guidelines. The following
agents were tested: ampicillin, cefazolin, erythromycin, gentamicin,
tetracycline and vancomycin (all from Sigma Chemical Co., USA),
teicoplanin (Aventis Pharma, France), ceftizoxime (Astellas Pharma
Inc., Japan), ciprofloxacin (Bayer Yakuhin Co., Ltd, Japan) and
imipenem (Banyu Pharmaceutical Co., Ltd, Japan). S. aureus ATCC
29213 was used as a reference strain.

Staphylocoagulase (SC) typing

Nucleotide sequencing of novel SC genes

Determination of the SC genes (coa) was performed by PCR
amplification and subsequently nucleotide sequencing the genes as
described previously.19

Genetic analysis
Multiple alignments were performed with GENETYX-MAC
Version 13.0.3 (GENETYX Corporation). The phylogenetic tree
was created using the neighbour-joining method in GENETYXMAC. Support for individual nodes on the tree was determined by
bootstrap analysis.

Multilocus sequence typing (MLST)

MLST was performed with primer sequences developed by Enright
et al.20 and Crisostomo et al.21 using real-time LightCycler System
PCR and amplification of the seven housekeeping genes in the same
PCR program.10 The STs were grouped into clonal complexes (CCs)
and shared a common ancestor as predicted by the based upon
related STs (eBURST) software v3.

spa typing

Materials and methods

spa typing was carried out according to Harmsen et al.22 and was
adapted to LightCycler System PCR with the SYBR Green I dye.
Samples contained 0.5 mM of forward primer, 0.3 mM of reverse
primer and 3 mM MgCl2 and were pre-incubated for 10 min at 958C
and then subjected to 35 cycles of amplification run according to
the following schedule: denaturation at 958C for 0 s, annealing at
558C for 5 s and extension at 728C for 18 s. Melting curve analysis
was conducted by continuously registering the fluorescence while
slowly raising the temperature (0.18C/s) from 658C to 958C. Prior to
sequencing, the products amplified by LightCycler System PCR
were purified using a MultiScreen PCRm96 Plate (Millipore AB,
Solna, Sweden) and subsequently sequenced on both strands using
the PCR primers and an ABI PRISM BigDye Terminator version
3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystems,
Stockholm, Sweden), and separation was performed on an ABI
PRISM 3100 Genetic Analyzer (Applied Biosystems). spa types

MRSA (n 16) that had previously been characterized as type IV

SCCmec by detection of the class B mec complex and the ccr type
2 genes using the scheme developed by Ito et al. and adjusted for
real-time LightCycler PCR (Roche Diagnostics, Mannheim,
Germany) and could not be further subtyped as IVa, IVb, IVc or IVd
were further analysed.4,17 The MRSA were isolated during 1983
2005, and outbreak isolates or isolates representing intrafamilial
spread were excluded. Thirteen of the isolates were classified as
community-acquired. Community-acquired infection was defined as
identification of MRSA in the outpatient setting or a culture positive
for MRSA within 48 h after hospital admission. In addition, the
patient had no medical history of MRSA infection or colonization
and no medical history in the last year of hospitalization, admission
to a nursing home, dialysis or surgery. The patient had no permanent
indwelling catheters or medical devices that pass through the skin.

33

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The SC type (I VIII) was determined by multiplex PCR to detect

types III, IV, VII and VIII (MPCR:A) and types I, II, V and VI
(MPCR:B) separately, as described previously.18

Berglund et al.
Table 1. Oligonucleotide primers used for long-range PCR and amplification of type IV SCCmec
Primer

Nucleotide sequence 50 !30

Location

Reference

cR2
cR1
mR3
mD2
mD3
mA9
mA2
IS7
IS8
a7
a5
cLm1
cL5

AAACGACATGAAAATCACCAT
AAGAATTGAACCAACGCATGA
CGCATCATTTATGATATGCTTCT
ATCACATTAATCGCACTTTTA
ACGCATTCTTACTGAGATTA
GTTGGTCCCATTAACTCTGAA
AACGTTGTAACCACCCCAAGA
ATGCTTAATGATAGCATCCGAATG
TATGTAGTATATCGAGCTTCACA
ACAAGTCATAGGCTATTTACGT
AGGACAGACGTAGTAACGTAATGT
AGCTAAAACTTTGCTTCACTATA
AGCAGTTCATAATTGTCATCA

orfX
orfX
extremity of SCCmec
hypervariable region
hypervariable region
mecA
mecA
IS1272
IS1272
ccr 2
ccr 2
right chromosomal region
right chromosomal region

Ito et al. 26
Ito et al. 26
this study
this study
this study
this study
Ito et al.17
Kondo et al.25
this study
this study
Ma et al.27
this study
this study

by nucleotide determination of the J1 region. Two isolates,

JCSC6668 and JCSC6670, were possible to analyse by PCR amplification from the ccr genes to the right chromosomal region. Other
isolates were not possible to amplify due to unknown chromosomal
regions or were designated as type IVg or IVh SCCmec. The entire
nucleotide sequence was determined of two SCCmec from isolates
JCSC6668 and JCSC6670. The SCCmec of isolates JCSC6668 and
JCSC6670 were amplified by using overlapping primers and several
long-range PCRs carried out with the Expand High Fidelity PCR
System (Roche Diagnostics) to cover the entire cassettes. The region
from orfX to the hypervariable region (HVR) was amplified using
primers cR1 and mD2 and from HVR to mecA using primers mD3
and mA9.26 The mec complex was covered using primer mA2
located in mecA and IS7 located in IS1272, and the region from the
mec complex to the ccr genes was amplified using primers IS8 and
a7.17,25 The region from the ccr genes to the chromosomal region
was amplified using primer a5 located in ccr and in the right chromosomal region the primer cLm1 was used for isolate JCSC6668
while primer cL5 was used for JCSC6670.27 Additional
primers (cR2 and mR3) were used to cover the entire orfX.26
Oligonucleotide primers used for long-range PCR amplification of
SCCmec are listed in Table 1, and the locations are indicated in
Figure 1. The PCR products were purified using the High Pure PCR
Purification Kit (Roche Diagnostics GmbH) and subsequently
sequenced on both strands using the ABI PRISM BigDye
Terminator version 3.1 Ready Reaction Cycle Sequencing Kit
(Applied Biosystems, Warrington, UK), and separation was performed on an ABI PRISM 3100 Genetic Analyzer (Applied
Biosystems). The sequences were analysed and assembled using the
BioEdit Sequence Alignment Editor.
orfs of more than 100 bp were identified with the
GenomeGambler v.1.5 software and were compared with sequence
databases at the National Center for Biotechnology Information with
the basic local alignment search tool (BLAST; National Library of
Medicine, Bethesda, MD, USA) for annotation and prediction of
functions. The ccr genes were investigated using the clustal W
(1.83) multiple alignment, and amino acid identities were calculated
using the EMBOSS pairwise alignment algorithms.

Detection of lukS-PV and lukF-PV

A previously described protocol was used to detect the Panton
Valentine leucocidin (PVL) genes employing LightCyclerw
FastStart DNA MasterPLUS SYBR Green I and modified regarding
the PCR conditions.23,24

Typing of SCCmec
SCCmec typing was initially performed by using real-time
LightCyclerw System PCR to detect the essential genetic components
mecA, mecR1, IS1272, ccrA and ccrB according to Berglund et al.16
and was confirmed by the multiplex scheme described by Kondo
et al.25 In the latter scheme, six multiplex PCRs identified the ccr gene
complex, the mec gene complex and specific structures in the J regions.

Detection of type IVg and IVh SCCmec

Primers designed in the J1 region of the isolate M03-68
(DQ106887), IV g Left 50 -GCAAGCTGTTATCGGCATTT-30 and
IV g Right 50 -GATCGTTCGTGTTTGTGTGC-30 , were used to
detect type IVg SCCmec and yielded a product of a size of 378 bp.7
The PCR was run at 948C for 2 min followed by 30 cycles of denaturation at 948C for 30 s, annealing at 608C for 1 min and extension
at 728C for 1 min, followed by a final extension at 728C for 2 min
before cooling at 48C.
Type IVh SCCmec was detected by employing primers J IVh
F 50 -TTCCTCGTTTTTTCTGAACG-30 and J IVh R 50 -CAAAC
ACTGATATTGTGTCG-30 .9 The PCR was run at 948C for 1 min
followed by 30 cycles of denaturation at 948C for 1 min, annealing
at 578C for 1 min and extension at 728C for 1 min, followed by a
final extension at 728C for 1 min before cooling at 48C.

Nucleotide sequencing of SCCmec

Nucleotide sequence accession numbers

Type IV SCCmec was initially identified by detection of a class B

mec complex and the type 2 ccr, and isolates carrying subtypes
other than IVa, IVb, IVc, IVd, IVg or IVh were further investigated

The sequence of the SCCmec elements of strains JCSC6668

(CCUG41764) and JCSC6670 (CCUG27050) have been deposited

34

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were determined with the Ridom StaphType software (Ridom

GmbH, Wurzburg, Germany) and grouped using the BURP (Based
Upon Repeat Pattern) algorithm.

MRSA with type IV SCCmec in Sweden

JCSC3063 type II.2 SCCmec

orfX

Tn554
mecA
mecR1 mecI

J3

ccrB2 ccrA2
J2

J1

JCSC6668 type IVi (IV.7) SCCmec

Class B.3 mec

orfX
cR1

IS431

mecA mecR1
ISSep1 IS1272

mD2 mA2
mD3
mA9

IS8

IS7

ccrB2 ccrA2
5

cLm1

JCSC6670 type IVj (IV.8) SCCmec

orfX

IS431

mecA mecR1
IS1272

ccrB2 ccrA2

mecR1 IS1272

ccrB1 ccrA1

cL5

PL72 type I.2 SCCmec

Figure 1. Genetic structures of SCCmec of strains JCSC6668 and JCSC6670 based on the nucleotide sequences deposited in the DDBJ/EMBL/GenBank
databases under accession numbers AB425823 and AB425824, respectively, and illustrative comparison with type I.2 and II.2 SCCmec of isolates PL72
(accession number AB433542) and JCSC3063 (accession number AB127982), respectively. Amino acid homology is indicated in light grey (mec gene
complex) or light orange (ccr gene complex). orfs in the J1 regions are coloured in pink or purple. In type I.2 and II.2 SCCmec, broken lines represent
non-investigated areas. Red arrows indicate DR sequences and black arrowheads indicate positions of primers used; these are also listed in Table 1.

ST140 or ST182) and carried either type IVg SCCmec, IVh

SCCmec or unknown subtypes of type IV SCCmec.
Five of the 16 MRSA were classified as subtype IVg
SCCmec, and these represented five different genotypes as
depicted by spa and MLST (ST1, ST30, ST45, ST59 or ST140).
In contrast, four of the MRSA were classified as subtype IVh
SCCmec, and these were all ST22. However, one of the MRSA
carrying type IVh SCCmec had a different, yet closely related,
spa type (t020) and carried the PVL locus, in comparison with
the other MRSA with type IVh SCCmec that represented t032
and were PVL-negative. In addition, isolate JCSC6668 was positive in SCCmec multiplex PCR for identifying S01 located at
the J1 region of type II.2 SCCmec, indicating the presence of
the J1 region similar to type II.2 SCCmec (data not shown).

in the DDBJ/EMBL/GenBank databases under accession numbers

AB425823 and AB425824, respectively. The sequences of coa of
S. aureus JCSC6074, JCSC6666, JCSC6075, JCSC6667, JCSC6671,
JCSC6674 and JCSC6669 strains have been deposited in the DDBJ/
EMBL/GenBank databases under accession numbers AB436965,
AB436966, AB436967, AB436968, AB436969, AB436970 and
AB436971, respectively.

Results
Characterization of Swedish MRSA with type IV SCCmec
In order to define the collection of MRSA isolated in Sweden
carrying type IV SCCmec, we determined the MLST, spa and
SC types of these strains. As shown in Table 2, the selection of
Swedish MRSA carrying type IV SCCmec demonstrated a high
diversity of genotypes. A heterogeneous pattern of MICs was
observed, and several isolates showed notable low values of
carbapenems and cephalosporins. Sixteen Swedish MRSA
strains were distributed into nine STs (as shown in Table 2), and
these were classified into seven CCs and two singletons.
spa typing revealed that there were 13 different spa types
among these 16 Swedish MRSA strains. Most of the spa types
(11 of 13) were represented by single isolates, whereas two spa
types (t015 and t032) were represented by at least two strains.
SC types were determined using multiplex PCRs.18 Eight of
16 strains were classified into four SC types (II, III, IV and VII),
whereas the other eight strains could not be characterized
according to SC types I VIII. SC type VII was the major SC
group in this study, which included five strains of MRSA ST1,
ST45 and ST59. The eight SC non-typeable strains were represented by four genotypes according to MLST (ST22, ST45,

Identification of novel type of SC

The structures of coa genes of the isolates that had unknown
SC types were investigated by nucleotide sequencing of the
genes and comparison with existing type I X coa genes. We
determined the nucleotide sequences of coa of the eight MRSA:
JCSC6674, JCSC6665, JCSC6666, JCSC6667, JCSC6669,
JCSC6671, JCSC6074 and JCSC6075. The coa genes of these
eight isolates were classified into four groups based on their
nucleotide sequences. The coa genes of four MRSA, JCSC6075,
JCSC6667, JCSC6671 and JCSC6674, were identical to each
other. We designated the coa type of these isolates as type XIa.
The nucleotide sequence of type XIa showed . 99% identity to
the partial sequence of coa of HinfI coagulase type D strain
RS54.28 Type D was one of the major clones of MRSA isolated
in Scotland in the 1990s, and the PFGE banding patterns of the
strains were related to EMRSA-15.28 The coa of JCSC6669
showed 98% identity to the partial sequence of coa of HinfI
35

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orfX

Table 2. Characteristics of the 16 MRSA with unknown subtypes of type IV SCCmec from Sweden
MIC (mg/L)f
Year Origina PVL spa b STc CC Coagulase ccr mec complex SCCmec d Subtypee VAN TEC AMP CFZ ZOX

JCSC6668
JCSC6670
JCSC6673
JCSC6674
JCSC6663
JCSC6664
JCSC6665
JCSC6666
JCSC6667
JCSC6669
JCSC6671
JCSC6068
JCSC6073
JCSC6074
JCSC6075
JCSC6076

1999
1990
2005
2005
1983
1990
1994
1998
1998
1999
1999
2001
2004
1995
1998
1999

C
C
C
C
C
C
C
C
C
C
C
H
H
C
H
C

2
2

2
2
2
2
2
2
2
2
2
2
2
2

t127
t002
t300
t020
t009
t216
t1231
t296
t032
t493
t032
t015
t015
t957
t032
t590

1
5
30
22
254
59
45
140
22
182
22
45
45
140
22
1

1
5
30
22
8
59
45

22

22
45
45

22
1

VII
II
IV
XIa
III
VII
VIIb
Xb
XIa
XIb
XIa
VII
VII
Xb
XIa
VII

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

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

IV/II.b
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV
IV

new
new
IVg
IVh
unknown
IVg
IVg
IVg
IVh
unknown
IVh
unknown
unknown
unknown
IVh
IVg

2
2
0.5
1
2
1
1
1
1
1
1
1
1
1
0.5
2

2
2
2
1
1
1
1
2
1
2
1
1
1
1
1
1

64
32
128
32
64
64
128
128
64
16
64
32
32
.128
64
64

64
64
256
256
2
32
128
64
.128
.128
.128
32
4
64
.128
32

.128
.128
128
128
32
.128
.128
.128
.128
.128
.128
.128
128
.128
.128
.128

ERY

CIP

GEN

.128
64
32
1
1
2
2
0.5
0.25
8 .256
256
.128
0.25 128
1
1
1
8
1
64
2
1
1
.128
64
32
2
0.25
1
.128
64
1
0.5
0.25
0.25
1
0.5
1
2
0.5
2
.128
64
1
8
0.5
2

IPM

TET

2
8
0.5
1
,0.03125
2
32
2
16
32
16
0.125
0.0625
2
16
1

.128
0.5
32
4
128
0.5
0.5
.128
.128
0.5
1
0.25
0.5
.128
1
128

C, community; H, hospital.
The material consisted of 13 spa types. These were further grouped into three clusters (cluster 1, t296 and t957; cluster 2, t127 and t590; cluster 3, t015 and t1231) and seven singletons by using the
BURP algorithm.
c
The STs were grouped into CCs and shared a common ancestor as predicted by the based upon related STs (BURST) software. The CCs were named after the ST of the predicted ancestral genotype,
which were further divided into seven CCs (1, 5, 8, 22, 30, 45 and 59) and two singletons. ST22 was the most frequent ST consisting of four isolates, three isolates were ST45, and two isolates were ST140
as well as ST1. The other STs were represented by only a single isolate.
d
The isolate JCSC6668 was designated as a type IV SCCmec, but was also positive in multiplex PCR for SA01, a component of the type II.b SCCmec.
e
Based on differences in the J1 region.
f
VAN, vancomycin; TEC, teicoplanin; AMP, ampicillin; CFZ, cefazolin; ZOX, ceftizoxime; ERY, erythromycin; CIP, ciprofloxacin; GEN, gentamicin; IPM, imipenem; TET, tetracycline.
b

Berglund et al.

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36

Isolate

MRSA with type IV SCCmec in Sweden

characterized as type IV SCCmec by the presence of type 2 ccr
and class B mec complex and were further investigated by longrange PCR and determination of the nucleotide sequence of the
J1 region. The J1 regions of JCSC6668 and JCSC6670 had high
homology to corresponding regions previously described in type
II.2 and I.2 SCCmec, respectively, and the entire nucleotide
sequences of the two elements were therefore determined in
order to properly describe the SCCmec.

coagulase type I* strain a48 isolated in Aberdeen, and it was

designated as type XIb. The coa of two isolates, JCSC6074 and
JCSC6666, were identical to each other. The nucleotide identity
between coa sequences except repeat sequences of these two
isolates and that of type X coa was 92.8%, and therefore, the
coa were designated as type Xb. The other non-typeable coa of
JCSC6665 showed 92.1% identity to the coa sequence without
repeat sequence of type VII coa. We designated it as type VIIb.
We investigated the phylogenetic relation among the new coa
of Swedish MRSA and the type I X coa by using those nucleotide sequences, except repeat sequences, which is shown in
Figure 2. The phylogenetic tree of coa also showed that Swedish
MRSA of type IV SCCmec demonstrated a high diversity.

Characteristics of the two SCCmec

Identification of novel SCCmec type IV

Seven MRSA remained as unknown type IV SCCmec as five
isolates were carrying type IVg SCCmec and four isolates were
classified as carrying type IVh SCCmec. Five of the seven
MRSA carrying novel subtypes of type IV SCCmec were not
possible to amplify by long-range PCR covering the J1 region.
In two isolates, JCSC6668 and JCSC6670, 12 kb PCR products were covering the J1 regions. These isolates were initially

Type V
99.80%

Type VIII

93.20%

JCSC6075
99.90%

JCSC6671

99.90%

JCSC6674

Type XIa

Type XIb

JCSC6669

97.20%
56.90%

JCSC6667

100.00%

Type IV
100.00%

59.70%

Type IX
Type II
Type III

99.80%

100.00%

JCSC6074
JCSC6666

100.00%

Type Xb

Type X

100.00%

Type I
100.00%

JCSC6665
100.00%

Type VIIb

Type VII

Type VI
Figure 2. Neighbour joining tree of coa of Swedish MRSA carrying type IV
SCCmec and SC type I X reference strains. Nucleotide sequences of coa
other than the repeat region and C-terminal sequence were used for
comparison. The SC reference strains are as follows: 104 (type I), N315
(type II), NCTC 8325 (type III), stp28 (type IV), No55 (type V), stp12 (type
IV), MW2 (type VII), Ku (type VIII), 17573 (type IX) and 19 (type X).

37

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The entire SCCmec elements were amplified in isolates

JCSC6668 and JCSC6670 by long-range PCR and were subsequently sequenced. The SCCmec of JCSC6668 was 22 534 bp
long and the SCCmec of JCSC6670 was 22 323 bp, and both
SCCmec were demarcated by integration site sequences (ISSs)
of SCCmec. Also, two characteristic direct repeated (DR)
regions were located at the upstream extremity of the two
SCCmec and one DR at the downstream region.
The orfs of the two type IV SCCmec are summarized in
Table 3. Both SCCmec investigated carried the class B mec
complex as determined by the presence of a truncated copy of
IS1272 located downstream of mecR1. However, the SCCmec of
JCSC6668 had an altered class B mec complex with an integration of a 1014 bp ISSep1-like transposase located upstream
of a 12 bp truncated IS1272. This novel mec complex consisting
of IS431/mecA/DmecR1/ISSep1/DIS1272 was regarded as a
variant class B mec complex designated as class B.3 (Figure 1).
The two SCCmec identified in JCSC6668 and JCSC6670
carried the type 2 ccr and surrounding orfs. The amino acid
homology of ccrA in JCSC6668 was 98.9% identical to ccrA
described in a type IVa SCCmec (accession number AB063172)
and in a Staphylococcus epidermidis (accession number
DQ514334). The ccrB was 1629 bp and had 99.6% identity to
the ccrB described in an isolate representing USA300 (accession
number CP000730), in a type IVc SCCmec (accession number
AB266532), among others. The ccrA of JCSC6670 had 100%
amino acid identity to ccrA described in S. aureus isolate ZH47
and the type IVE SCCmec (accession numbers AM292304 and
AJ810121), as well as in Staphylococcus warneri and S. epidermidis (accession numbers DQ225180 and DQ196433). The ccrB
of JCSC6670 was 21 bp longer than ccrB in JCSC6668 and had
100% identity to ccrB described in type IVa and IVd SCCmec
(accession numbers AB266531 and AB097677) as well as in
isolate ZH47.
The J1 region of isolate JCSC6668 was 3.3 kb and showed
99.2% to 99.4% identity to the corresponding orfs described in
the type II.2 SCCmec of strain JCSC3063 (accession number
AB127982). The length was equal in the type II.2 SCCmec and
consisted of two orfs representing hypothetical proteins with
unknown functions. In addition, the J2 region of JCSC6668 had
high homology to a corresponding region in type II.2 SCCmec
of strain JCSC3063, although the latter consisted of a much
larger region, due to insertion of, for example, Tn554.
The isolate JCSC6670 highly corresponded (99.4% to 100%
identity) to the J1 region described in the SCCmec type I.2
identified in strain PL72 (accession number AB433542) (H.
Xiao, T. Ito, F. Takeuchi, X. X. Ma, M. Takasu, Y. Uehara, D.
Oliveira, H. de Lencastre and K. Hiramatsu, unpublished results)
and was 4.7 kb. The J1 regions of both elements consisted of
four orfs of equal length, which were considered as hypothetical

Berglund et al.
Table 3. orfs in type IV SCCmec of JCSC6668 and JCSC6670 and comparison with type I.2 and II.2 SCCmec
Homology to ORF in
existing SCCmec b

Value for CDSa

ORF

location

gene size
(bp)

length
(aa)

4-483
805-2061
3421-2747 (complement)
4723-5466

480
1257
675
744

160
419
225
248

CB05
CB06
CB07

5563-5991
8043-6037 (complement)
8143-9129

429
2007
987

143
669
329

CB08

9032-9361

216

71

CB09
CB10
CB11
CB12
CB13
CB14
CB15
CB16
CB17
CB18
CB19

9812-10825
12433-10922 (complement)
13075-12569 (complement)
13401-13090 (complement)
13838-13488 (complement)
15988-14360 (complement)
17359-16010 (complement)
19380-17593 (complement)
19694-19380 (complement)
21420-19975 (complement)
22864-21920 (complement)

1014
1512
507
312
351
1629
1350
1788
315
1446
945

338
504
169
104
117
543
450
596
105
482
315

3-482
804-2060
3420-2746 (complement)
4722-5465

480
1257
675
744

160
419
225
248

C05
C06
C07

5562-5990
8042-6036 (complement)
8142-9128

429
2007
987

143
669
329

C08

9031-9360

216

71

C09
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20

10874-9351 (complement)
11525-11010 (complement)
11842-11531 (complement)
12279-11929 (complement)
14450-12801 (complement)
15800-14451 (complement)
17827-16034 (complement)
18138-17827 (complement)
18295-19878
19950-20882
21308-21685
22154-22651

1524
516
312
351
1650
1350
1794
312
1584
933
378
498

508
170
103
116
550
450
598
104
528
311
126
166

JCSC6670
C01
C02
C03
C04

Description of product

orfX

corresponding
%
identityc ORF [size (bp)]

conserved hypothetical protein, OrfX

hypothetical protein
tnp
transposase of IS431mec
glycerophosphoryl diester
phosphodiesterase
MaoC domain protein
mecA penicillin-binding protein 20 (PBP20 )
mecR1 truncated signal transducer protein
MecR1
hsdR truncated restriction-modification
system endonuclease
tnp
ISSep1-like transposase
tnp
transposase for IS1272
hypothetical protein
hypothetical protein
hypothetical protein
ccrB2 cassette chromosome recombinase B2
ccrA2 cassette chromosome recombinase A2
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein

ND
ND
ND
ND

ND
ND
ND
ND

ND
ND
ND

ND
ND
ND

ND

ND

ND
ND
92.3%
89.3%
88.8%
97.4%
96.7%
100%
100%
99.2%
99.4%

ND
ND
S09 (519)
S08 (312)
S07 (351)
S06 (1629)
S05 (1350)
S04 (1788)
S03 (315)
S02 (1560)
S01 (945)

orfX

ND
ND
ND
ND

ND
ND
ND
ND

ND
ND
100%

ND
ND
ND (987)

100%

ND (260)

100%
96.5%
85.4%
85.3%
82.0%
75.6%
88.4%
98.9%
99.4%
100%
100%
100%

ND (1524)
ND (516)
ND (312)
ND (351)
ccrB1 (1625)
ccrA1 (1350)
ND (1773)
ND (297)
ND (1584)
ND (933)
ND (378)
ND (498)

conserved hypothetical protein, OrfX

hypothetical protein
tnp
transposase of IS431mec
glycerophosphoryl diester
phosphodiesterase
MaoC domain protein
mecA penicillin-binding protein 20 (PBP20 )
mecR1 truncated signal transducer protein
MecR1
hsdR truncated restriction-modification
system endonuclease
tnp
transposase for IS1272
hypothetical protein
hypothetical protein
hypothetical protein
ccrB2 cassette chromosome recombinase B2
ccrA2 cassette chromosome recombinase A2
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein
hypothetical protein

ND, not determined.

a
CDS, coding sequence. Nucleotide positions are determined based upon nucleotide sequence deposited in the DDBJ/EMBL/GenBank databases under
accession numbers AB425823 (JCSC6668) and AB425824 (JCSC6670), respectively, and they correspond to the 50 to 30 direction.
b
JCSC6668 in comparison with type II.2 SCCmec of JCSC3063; JCSC6670 in comparison with type I.2 SCCmec of PL72.
c
Identity of the amino acid sequence to each ORF.

38

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JCSC6668
CB01
CB02
CB03
CB04

Gene

MRSA with type IV SCCmec in Sweden

proteins. Although the SCCmec of JCSC6670 and PL72 had
different ccr, the J2 region was also well conserved in these
different cassettes.
In contrast to the J1 region, the two investigated type IV
SCCmec were well conserved in the J3 and J2 regions. The J3
regions located from orfX to mecA were 99.9% identical in
JCSC6668 and JCSC6670 and contained both the downstream
constant region and the HVR commonly identified among type
IV SCCmec. In addition, this region was 99.9% similar to the
existing type IVa SCCmec in isolate CA05 (accession number
AB063172).27 The J2 regions were 99.6% identical among the
two SCCmec investigated and 99.5% or 99.9% identical to the
type IVa SCCmec of CA05.

Discussion
We describe two new variants of type IV SCCmec in a collection of Swedish MRSA constituting type IV SCCmec with
unknown subtypes, i.e. not type IVa, IVb, IVc or IVd. The type
IV SCCmec of JCSC6668 and JCSC6670 were investigated by
nucleotide sequencing and were found to possess J1 regions
with high identity to J1 regions previously identified in type II.2
and I.2 SCCmec, respectively. High amino acid identities were
also observed in the J2 regions. The finding of equal J1 regions
explains why the isolate JCSC6668 was positive in the multiplex
PCR for S01, which would have indicated the presence of a type
II SCCmec according to the multiplex PCR described by Kondo
et al.25 Although this specific finding of equal J1 regions is
unique, difficulties in assigning SCCmec due to similarities in J
regions have been reported previously.25 Furthermore, variation
has recently also been observed in type II SCCmec, which
carries type 2 ccr genes as does type IV SCCmec.12,25,29,30
The occurrence of diverse J1 regions among type IV
SCCmec from Sweden indicated independent insertions of mec
complex into the J1 regions that may have served as the original
SCC. The SCCmec is suggested to initially have originated by
integration of a mec gene complex into the J1 region located
between the ccr genes and the downstream chromosomal
region.25 In this study, we identified nearly identical J1 regions
in different types of SCCmec, a finding that provides further
support for this hypothesis. However, no ISSs were identified
inside the SCCmec that would suggest an organization of two
preceding SCC elements. It is possible that the type IV SCCmec
described in this study shared a common origin with the type
II.2 and I.2 SCCmec, respectively, and that these elements may
have arisen by independent integration of the mec complex.
No type IV SCCmec have yet been described to carry these
compositions of the J1 region and, for that reason, we designate
the two new subtypes as types IVi SCCmec (JCSC6668) and
IVj SCCmec (JCSC6670), or IV.7 and IV.8 according to the
nomenclature recently suggested by Chongtrakool et al.8 MRSA
from the whole world are continuously being investigated and,
consequently, more SCCmec variants are being identified. This
new information gives us an idea of the origin and relatedness of
SCCmec.
The size of the type IV SCCmec of MRSA is more variable
and the composition more diverse than among the other
SCCmec types. There are until today six known subtypes of type
IV SCCmec based on structural differences in the J1 region
located between the ccr complex and the right chromosomal

Acknowledgements
hren, Christina Welinder-Olsson and Leif
We thank Christina A
Larsson, at the Sahlgrenska University Hospital, Gothenburg,
39

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region of SCCmec. Type IVa was described and frequently

found in CA-MRSA from the USA and Australia and has then
been identified all over the world, including Sweden.4,16,27,31,32
Type IVb has been scarcely found since it was first described in
the USA, which is in contrast to subtype IVc that is present in
MRSA spread all over Europe as one of the dominating
clones.4,5,16,33,34 Types IVc and IVd were frequently found in
MRSA isolated in Japan in the early 1980s.6 Subtype IVg was
found in MRSA from bovine milk in Korea.7 In the present
study, we describe MRSA with type IVg SCCmec isolated from
humans, and they all displayed different genetic backgrounds as
depicted by MLST, indicating independent introduction of the
SCCmec into the chromosome of S. aureus. The most recently
described subtype, IVh, was specific for EMRSA-15 and
reported to be highly homologous to the strain PL72, although
the entire SCCmec was not investigated.9 Four of the Swedish
MRSA carried type IVh SCCmec with the same genetic background, ST22, as the Portuguese MRSA with subtype IVh.
Interestingly, one of the MRSA with type IVh SCCmec carried
the PVL locus, which may indicate the introduction of the PVL
genes subsequent to the integration of the SCCmec.
The collection of 16 isolates further analysed in the present
study represented highly diversified MRSA designated as type
IV SCCmec, which consisted of at least nine different genetic
backgrounds as depicted by MLST, SC typing and spa typing.
The concordance between these three methods was high,
although the SCCmec showed a more complex picture. The
occurrence of subtype IVg, new subtypes and unknown subtypes
in the same genotype indicated a high level of recombination or
represented highly mobile type IV SCCmec. Type IV SCCmec
is smaller in size than the other SCCmec types; it is found
among S. aureus with several different genetic backgrounds and
generally does not contain any additional resistance genes,
which may facilitate mobility of the cassette. The larger size of
type II SCCmec is thought to be the explanation for the observation that this element is not excised by the ccr recombinases
and not readily spread among several genetic backgrounds.35 In
addition, the presence of four new SC types in the collection of
Swedish MRSA further indicated a diverse nature. The similarity
of XIa and XIb to MRSA strains described in Scotland probably
represents the clonal spread of S. aureus in Europe.28
This study provides new information about the variation of
type IV SCCmec and how these elements may have arisen and
spread by site-specific excision driven by the type 2 ccr genes.
In order to more easily designate the increasing number of
SCCmec variants, we recommend the use of the alternative
nomenclature based on ccr type and mec class as well as also
including the description of J regions indicated by numbers
representing differences in the J1, J2 and J3 regions.8
In conclusion, type IV SCCmec is occurring in heterogeneous
clones of MRSA in Sweden, and it seems that type IV SCCmec
itself is a highly diversified genetic element. We describe two
novel subtypes of type IV SCCmec with common J1 regions
shared by other types of SCCmec, which indicate that J1 regions
occurred as primordial SCC.

Berglund et al.

Funding
This study was supported by grants from the Sweden Japan
Foundation, the Swedish Society for Medical Research, the
rebro
Swedish Institute of Biomedical Laboratory Science, the O
County Council Research Committee, Sweden, and a
Grant-in-Aid for 21st Century COE Research, Ministry of
Education and Science, Japan.

Transparency declarations
None to declare.

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Sweden, for providing MRSA isolates. Tadashi Baba at the

Department of Microbiology and Infection Control Science,
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Department of Infection Control Science, Juntendo University
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41