JAC Advance Access originally published online on November 7, 2007
Journal of Antimicrobial Chemotherapy 2008 61(1):73-77; doi:10.1093/jac/dkm422
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Original research |
Distribution of the ACME-arcA gene among methicillin-resistant Staphylococcus aureus from England and Wales
Staphylococcus Reference Laboratory (SRL), Laboratory of Healthcare Associated Infection, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK
* Corresponding author. Tel +44-20-8327-7259; Fax: +44-20-8200-7449; E-mail: matthew.ellington{at}hpa.org.uk
Received 3 August 2007; returned 25 September 2007; revised 10 October 2007; accepted 11 October 2007
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Abstract |
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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 = 203) of diverse genetic types, referred to the England and Wales Staphylococcus reference laboratory, were tested for the presence of the ACME-arcA gene. ACME-arcA-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-MRSA-SCCmecIVa 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
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Introduction |
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Although epidemic healthcare-associated (HA)-methicillin-resistant Staphylococcus aureus (MRSA) strains 15 (ST22-MRSA-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
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 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.
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Materials and methods |
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Bacterial isolates
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 = 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.
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 5'-cgatatcatctatacctagtacg-3'/arcAchrRev 5'-gaaaatcctcaagtaagaagtg-3' was suitable to amplify native arcA. Reactions were performed essentially as described previously,8 using 0.1 µM each primer and cycling as follows: 5 min at 95°C, followed by 30 cycles of 95°C for 1 min, 50°C for 1 min and 72°C for 2 min, followed by 72°C 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 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 StaphType® 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
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
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
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Results |
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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.
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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 β-lactams; susceptibility to aminoglycosides, tetracycline, ciprofloxacin and erythromycin was variable (Table 1).
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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 β-lactams, 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 ACME-arcA-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 β-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.
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Discussion |
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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 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.
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This work was supported by the Health Protection Agency.
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None to declare.
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Acknowledgements |
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This work was presented at the Seventeenth European Congress of Clinical Microbiology and Infectious Diseases and at the Twenty-fifth International Congress of Chemotherapy, Munich, Germany, 2007.
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References |
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