Journal of Antimicrobial Chemotherapy (2008) 61, 73– 77
doi:10.1093/jac/dkm422
Advance Access publication 7 November 2007
Distribution of the ACME-arcA gene among methicillin-resistant
Staphylococcus aureus from England and Wales
Matthew J. Ellington*, Lianne Yearwood, Mark Ganner, Claire East and Angela M. Kearns
Staphylococcus Reference Laboratory (SRL), Laboratory of Healthcare Associated Infection, Centre for Infections,
Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK
Received 3 August 2007; returned 25 September 2007; revised 10 October 2007; accepted 11 October 2007
Background: The ST8-SCCmecIVa (USA300) methicillin-resistant Staphylococcus aureus (MRSA) clone
can harbour the arginine catabolic mobile element (ACME). The arc gene cluster within the ACME may
function as a virulence or strain survival factor. We determined the distribution of the ACME-associated arcA gene among genetically diverse MRSA from around England and Wales.
Methods: MRSA isolates (n 5 203) of diverse genetic types, referred to the England and Wales
Staphylococcus reference laboratory, were tested for the presence of the ACME-arcA gene. ACMEarcA-positive isolates were characterized by toxin gene profiling, PFGE and spa sequence typing.
MICs of a range of antimicrobials were also determined.
Results: The ACME-arcA gene was detected in 17 isolates. Twelve were related to known ST8-MRSASCCmecIVa isolates of the USA300 lineage by pulsotype and were resistant to oxacillin, with variable
ciprofloxacin and erythromycin resistance. Outside the USA300 lineage, four of the remaining five
ACME-arcA isolates were closely related ST97-MRSA-SCCmecV, Panton-Valentine leucocidin (PVL)negative, resistant to oxacillin and variously resistant to erythromycin, ciprofloxacin, clindamycin,
gentamicin, tetracycline and fusidic acid. The remaining isolate was ST1, PVL-positive and resistant to
fusidic acid as well as oxacillin. Thirteen out of the 17 isolates were associated with skin and soft
tissue infections.
Conclusions: The detection of ACME-arcA in diverse MRSA types highlights the mobility of the
elements encoding ACME-arcA genes. The diversity of strain types and resistance profiles among
ACME-arcA-encoding MRSA is a cause for public-health concern and demands continued surveillance
and close monitoring.
Keywords: USA300, pathogenicity, virulence, community
Whole genome sequencing of a USA300 strain, with the
0114 pulsotype, revealed the presence of a novel acquired
genetic element named the arginine catabolic mobile element I
(ACME I). The element had a structure similar to that of staphylococcal chromosomal cassettes. Although the element did not
encode a ccr-like recombinase, ACME I had inserted into the
orfX gene adjacent to the SCCmecIVa cassette.5 Although
unproven, ACME I has been suggested to contribute to the
success of the USA300 strain through enhancing virulence and/
or fitness.5 The element encodes an additional copy of the arginine catabolism gene cluster arcRADBC, in addition to other
factors such as the oligopeptide permease (opp3) encoding gene
cluster. The ACME-encoded arcA gene is distinct from the
S. aureus arcA housekeeping gene and can be detected by PCR
Introduction
Although epidemic healthcare-associated (HA)-methicillinresistant Staphylococcus aureus (MRSA) strains 15 (ST22MRSA-SCCmecIV) and 16 (ST36-MRSA-SCCmecII) remain
predominant in the UK, new MRSA lineages have been
detected.1 Among these is the multiple locus sequence type
(MLST) 8 (ST8), SCCmecIVa, PFGE type USA300 clone. This
strain type has caused infections in various communities2 as well
as in healthcare patients and has been reported as existing alongside and even displacing traditional epidemic MRSA types
associated with the healthcare setting in the USA.3 Specifically,
this clone is often associated with skin and soft tissue infections,2
but can cause other disease pathologies such as bacteraemia.4
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
*Corresponding author. Tel þ44-20-8327-7259; Fax: þ44-20-8200-7449; E-mail: [email protected]
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
73
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Ellington et al.
using primers specific for the acquired-ACME-arcA variant.5 To
date, the ACME-associated arcA gene has been reported predominantly in the ST8-SCCmecIVa (USA300-0114) genetic
background.5,6 Recently, it has also been detected in ST8
USA300-methicillin-susceptible S. aureus (MSSA) and ST5,
USA100-MRSA-SCCmecII.6 We have determined the distribution of ACME-arcA among genetically diverse isolates
of community-associated (CA)- and healthcare-associated
(HA)-MRSA in England and Wales. Here, we present the detection of ACME-arcA in UK isolates of the ST8 SCCmecIVa
(USA300) strain and report for the first time (to our knowledge)
its presence in isolates of ST97 SCCmecV and ST1 SCCmecIVa
MRSA.
sequenced on both strands with a Beckmann CEQ8000 DNA
sequencer (Beckmann Coulter, High Wycombe, UK) according to
the manufacturer’s instructions. Data were analysed using Ridom
StaphTypew software (Ridom GmbH, Wurzburg, Germany).
Multilocus sequence typing was performed as described
previously.10
Pulsed-field gel electrophoresis
Strain relatedness was analysed by PFGE of total DNA restricted
with SmaI, as described previously.8 Banding patterns were analysed
using BioNumerics software (Applied Maths, Ghent, Belgium), and
isolates with profiles .80% similar were considered closely related.
SCCmec cassette, ccr typing and accessory gene regulator
(agr) allotyping
Materials and methods
Bacterial isolates
SCCmec cassette identification was carried out by PCR using previously described PCR schemes,11 – 13 and the ccr type of the
ACME-arcA-positive isolates was determined.8,14 PCR was used to
determine the agr allotype.8
The England and Wales Staphylococcus reference laboratory
receives isolates from diverse infection types for epidemiological
typing and investigation. We selected 203 contemporaneous, genetically diverse isolates including representatives of HA and CA types
in order to determine the distribution of the ACME-arcA gene
among diverse isolates from around England and Wales. The isolates tested included 12 PFGE subtypes each of EMRSA-15
(ST22-MRSA-SCCmecIV) and -16 (ST36-MRSA-SCCmecII) as
well as genetically diverse CA-MRSA types, identified on the basis
of SCCmec type, and PFGE clustering with previously MLST and
spa-defined isolates. These included predicted ST1-IVa, ST5-IV and
IVc, ST8-IVa, ST22-IVA, ST30-IVc and ST80-IVc isolates (n ¼ 16,
52, 36, 13, 9 and 36, respectively), one isolate each of ST59-V and
ST88-IVa and 15 isolates with SCCmecIV but of undetermined ST.
Patient demographic and clinical data for ACME-arcA-positive
isolates were collated where possible.
Toxin gene profiling
Isolates were further characterized by four different multiplex PCRs
to detect genes for staphylococcal enterotoxins A –E and G– J, toxic
shock syndrome toxin-1, exfoliative toxins A, B and D, and
Panton-Valentine leucocidin (PVL), with each reaction containing
primers for the 16S rRNA gene as an internal control.8
Results
The ACME-arcA gene was detected in 17 of 203 MRSA isolates; these were identified in 10 centres around England.
Thirteen of 17 ACME-arcA-positive isolates were obtained from
skin and soft tissue infections (Table 1), two isolates were from
screening swabs, one from a urinary tract infection, and the isolation site of one isolate was unknown. Six isolates were from
patients after .48 h of hospital care, 10 were isolated from individuals attending outpatient clinics, and 1 isolate was of
unknown origin.
Twelve of 17 ACME-arcA-positive isolates were spa type
t008 (Table 1) and were closely related by pulsotype to a confirmed ST8 control isolate (Figure 1); four were confirmed as
ST8 by MLST. All 12 isolates harboured the SCCmecIVa
element, were PVL-positive, agr allotype 1 and ccr type A2
(Table 1). These isolates were resistant to b-lactams; susceptibility to aminoglycosides, tetracycline, ciprofloxacin and
erythromycin was variable (Table 1).
A further four ACME-arcA-positive MRSA were closely
related by spa sequence typing, with three being t359 and one
differing by a single r34 repeat duplication (spa type t267). One
representative t359 isolate and the t267 isolate were confirmed
as MLST ST97 by DNA sequencing (Table 1 and Figure 1).
PFGE confirmed that these isolates were closely related: the
three t359 isolates were indistinguishable by PFGE and the t267
isolate was .80% similar based on PFGE banding patterns. All
four isolates were agr type 1, harboured the SCCmecV element,
ccrC, genes encoding staphylococcal enterotoxins D and J, and
were PVL-negative (Table 1). They were resistant to b-lactams,
Susceptibility testing
MICs were determined by Etest (AB BIODISK, Solna, Sweden) or
agar dilution using Iso-Sensitest agar (Oxoid, Basingstoke, UK),
according to the BSAC method.7 The antimicrobials tested were
penicillin, oxacillin, ciprofloxacin, tetracycline, erythromycin, gentamicin, fusidic acid, clindamycin, rifampicin, teicoplanin and
vancomycin.
Detection of the ACME and native arcA genes
An alignment of the USA300 acquired-ACME and native S. aureus
arcA genes (GenBank accession numbers: YP_492784 and
YP_495204) suggested that previously described primers were suitable to screen isolates for ACME-arcA,6 whereas the primer pair
arcAchrFwd 50 -cgatatcatctatacctagtacg-30 /arcAchrRev 50 -gaaaatcct
caagtaagaagtg-30 was suitable to amplify native arcA. Reactions
were performed essentially as described previously,8 using 0.1 mM
each primer and cycling as follows: 5 min at 958C, followed by 30
cycles of 958C for 1 min, 508C for 1 min and 728C for 2 min,
followed by 728C for 5 min.
Sequence-based typing and DNA sequencing
Single-locus spa sequence typing was carried out.9 DNA sequencing
template was prepared using EXOSAP.IT (GE Healthcare, Little
Chalfont, UK) according to the manufacturer’s instructions, and
74
Table 1. Characteristics of MRSA isolates harbouring the ACME-associated arcA gene
MIC (mg/L)
ST
type
spa
agr
type
ccr
type
Toxin gene
profile
8
8
8
8a
8a
A
A
A
A
A
t008
t008
t008
t008
t008
1
1
1
1
1
A2
A2
A2
A2
A2
lukSF-PV
lukSF-PV
lukSF-PV
lukSF-PV
lukSF-PV
IVa
IVa
IVa
IVa
IVa
8
A
t008
1
A2
lukSF-PV
IVa
abscess
abscess
abscess
nasal swab
wound
infection
boils
8a
A
t008
1
A2
lukSF-PV
IVa
blisters
8a
8a
A
A
t008
t008
1
1
A2
A2
lukSF-PV
lukSF-PV
IVa
IVa
abscess
abscess
8a
A
t008
1
A2
lukSF-PV
IVa
abscess
a
8
8a
A
A
t008
t008
1
1
A2
A2
lukSF-PV
lukSF-PV
IVa
IVa
nasal swab
UTI
1
B
t127 3
A2
IVa
abscess
97
97a
97
97a
C
C
C
C
t267
t359
t359
t359
sea, seh,
lukSF-PV
sed, sej
sed, sej
sed, sej
sed, sej
V
V
V
V
cellulitis
NA
wound swab
cellulitis
1
1
1
1
C
C
C
C
SCCmec
Disease
group
lukSF-PV¼Panton-Valentine leucocidin.
sea, sed, sej, seh¼staphylococcal enterotoxins A, D, J and H, respectively.
a
MLST type inferred from related isolates.
fusidic
acid
Patient location oxacillin penicillin ciprofloxacin erythromycin linezolid trimethoprim gentamicin tetracycline vancomycin
GUM clinic
GUM clinic
paediatric
inpatient
general
practitioner
general
practitioner
general
practitioner
outpatient
general
practitioner
general
practitioner
inpatient
general
practitioner
inpatient
inpatient
NA
inpatient
general
practitioner
128
128
128
128
128
64
64
64
64
64
16
16
0.5
0.5
0.5
0.5
0.5
64
64
64
2
2
2
2
2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
1
64
64
0.25
0.25
0.25
1
1
1
1
1
0.125
0.125
0.125
0.125
0.125
128
64
0.5
64
2
0.5
8
0.25
2
0.125
64
64
8
64
2
0.5
1
0.25
1
0.125
32
128
64
64
0.25
1
64
2
2
2
0.5
0.5
1
0.5
64
0.25
1
1
0.125
0.125
128
64
0.5
64
2
0.5
0.5
0.5
1
0.125
32
128
64
64
0.25
0.5
64
64
2
2
0.5
0.5
1
1
64
0.25
1
1
0.125
0.125
32
32
0.5
0.5
2
1
1
0.5
1
8
4
4
4
4
2
2
2
2
1
0.75
1
1
.128
0.5
0.5
0.5
2
2
2
2
8
16
16
16
0.5
0.5
0.5
0.5
0.25
0.25
0.25
0.25
1
1
1
1
0.125
0.125
0.125
0.125
ACME-arcA in diverse MRSA
75
PFGE
profile
Ellington et al.
ST97-MRSA in the UK and ST97-MRSA-SCCmecV (with
ccrC) internationally and is the first time that the ACME-arcA
gene has been found in isolates outside CC5 and CC8. The
amplification of two sequence-distinct fragments of the arcA
gene from these newly identified ACME-arcA-positive lineages
supports the notion that these isolates may, like USA300-0114,
have a copy of the ACME-arcA gene in addition to the chromosomal S. aureus arcA gene. In the USA300 strain, the
ACME-arcA gene and its gene cluster are suggested to contribute to strain survival on the skin and/or function as a pathogenicity factor that influences skin infection.13 Notably, other
factors within ACME elements—such as the opp3 gene cluster
(ACME I)—have been shown to have a virulence function and
to interact with arc-encoded functions.16 Also, other factors
outside the ACME-arc-encoding DNA elements may affect
strain virulence. Notwithstanding, 13 of the 17 isolates with the
ACME-arcA gene identified in this study were associated with
skin and soft tissue infections.
The ST1 isolate differed from other ACME-arcA isolates by
PFGE, spa and SCCmec typing and harboured genes for different superantigenic toxins. The isolate did bear some resemblance
by PFGE to representatives of the USA400 clone, previously
described as associated with a cluster of skin and soft
tissue infections among paediatric and maternity patients in
a New York City hospital.17 However, a recent survey of S.
aureus isolated in the USA did not identify any
ACME-arcA-positive isolates similar to USA400.6 The four
ST97 isolates identified in this study were clonal by PFGE and
harboured the SCCmecV element and the genes encoding the
staphylococcal enterotoxins D and J. Further, we report the first
finding of ACME-arcA in an SCCmecV-positive clone. To date,
ACME-arcA has been associated with the SCCmecIVa or II
elements.6 Although we did not identify the ACME-arcA gene
in isolates containing SCCmec elements I, II, III, IVb, c, d, it
should be noted that the isolates screened included representatives of each type and did not constitute a comprehensive collection of isolates from the UK. The presence of ACME-arcA
in strains of diverse genetic backgrounds suggests that
ACME-arcA-encoding element(s) may be highly mobile.
Moreover, the arc gene cluster in ACME I is flanked by transposases,5 and it is conceivable that the ACME-arcA amplicons
from the ST97 and ST1 isolates may not be associated with
complete ACME-like element(s), but could be present in these
strain types as a result of transposition event(s). This and other
investigations for these ACME-arcA types will form the subject
of future work.
To the best of our knowledge, this is the first report of
ACME-arcA in genetic backgrounds of MRSA other than
USA300 within Europe and, furthermore, is the first description
of the ACME-arcA gene not only in representatives of CC1 and
CC97, but also in SCCmecV-positive isolates. The emergence of
CA-MRSA strains harbouring markers for acquired putative
pathogenicity/strain transmissibility/fitness factors is a matter
demanding detailed study in conjunction with close monitoring
and surveillance internationally.
Figure 1. SmaI PFGE profiles of ACME-positive isolates. Lane 1 shows a
molecular weight marker. Lanes 2–13 show banding patterns of
ACME-arcA-positive ST8-like isolates, including four confirmed by MLST.
Lane 14 shows an ACME-arcA-negative confirmed ST8 isolate. Lane 15
shows the MLST-confirmed ST1 ACME-arcA isolate. Lanes 16–19 show
ACME-arcA-positive ST97 isolates including two MLST-confirmed isolates.
Letters on the right-hand side denote clusters of isolates with PFGE patterns
related at 80% or above.
ciprofloxacin and trimethoprim; resistance to erythromycin was
variable (Table 1).
On the basis of the spa type and the PFGE banding pattern, a
single ST-1 isolate was distinct from any other ACMEarcA-positive isolate (Figure 1). In addition, this isolate was
distinct from other ACME-arcA isolates by spa typing (t127),
was agr allotype 3, harboured SCCmecIVa and ccr type A2, was
positive for the genes encoding PVL and the staphylococcal
enterotoxins A and H, and (other than b-lactams) was resistant
to fusidic acid.
Comparison of DNA sequence data from ACME-arcA and
native arcA gene fragments showed two distinct sequence types
to be present in each of the ST8, ST1 and ST97 isolates. The
ACME-arcA sequences from the ST8-SCCmecIVa representative
isolates were identical to their equivalent sequences in the
USA300 genome sequence. The fragments of ACME-arcA
amplified from the four ST97 isolates and the ST1 isolate were
identical to each other and were 99% identical to the equivalent
sequence from the USA300 genome sequence, with a single C-T
synonymous nucleotide substitution (GenBank accession
number: EF589677) versus the USA300 GenBank sequence.
These observations indicate relatively high levels of sequence
conservation within the most variable region of the acquired
ACME-arcA gene in these isolates.
Discussion
To date, the ACME-arcA gene has been identified in S. aureus
belonging to clonal complexes CC5 and CC8 in the USA.
Among isolates from the UK, we found the ACME-arcA gene in
CC8 (equivalent to USA300) as well as representatives of CC97
and CC1 MRSA strains. ST97 MSSA have been reported in
many countries, and ST97-MRSA-SCCmecIV have been
reported in the USA.15 However, this is the first report of
Acknowledgements
This work was presented at the Seventeenth European Congress
of Clinical Microbiology and Infectious Diseases and at the
76
ACME-arcA in diverse MRSA
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Funding
This work was supported by the Health Protection Agency.
Transparency declarations
None to declare.
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