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J Clin Microbiol. Jun 2007; 45(6): 1981–1984.
Published online Apr 4, 2007. doi:  10.1128/JCM.00273-07
PMCID: PMC1933090

Epidemiologic Distribution of the Arginine Catabolic Mobile Element among Selected Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates[down-pointing small open triangle]

Abstract

We tested 214 Staphylococcus aureus isolates for the arcA locus of the arginine catabolic mobile element (ACME). All USA300 SCCmec IVa isolates, but no isolates containing other SCCmec subtypes, were arcA positive. arcA was also detected in selected methicillin-susceptible USA300 and methicillin-resistant USA100 isolates. DNA sequence analysis confirmed the integration of ACME in orfX.

Staphylococcus aureus continues to be an important human pathogen in both health care and community settings (11, 16). Methicillin-resistant S. aureus (MRSA) isolates of multilocus sequence type (MLST) clonal complex 8 (CC8), sequence type 8 (ST8), and pulsed-field gel electrophoresis (PFGE) type USA300 (14) are especially problematic, causing infections in a variety of community-associated populations (2, 10, 20, 21). USA300 isolates have also been reported among hospitalized patients (19) and patients with skin and soft tissue infections presenting to emergency departments across the United States (15).

Recent DNA sequence analysis of the USA300-0114 genome identified a novel genetic region, designated the arginine catabolic mobile element (ACME), that potentially enhances virulence and the ability to colonize humans (5). ACME integrates into orfX, the attachment site for the staphylococcal cassette chromosome (SCC) mec element, suggesting that ACME integration and excision could be catalyzed by the SCCmec recombinase. The ACME arcA gene was detected in USA300 isolates but not in other CC8:ST8 S. aureus strains or PFGE types (5). The goal of this study was to explore ACME distribution in additional USA300 isolates, including methicillin-susceptible S. aureus (MSSA) strains, and in S. aureus strains of other USA types and lineages.

A total of 214 S. aureus isolates from the strain collections of the Centers for Disease Control and Prevention (CDC) and Creighton University were selected for testing on the basis of their PFGE types, SCCmec profiles, and antimicrobial resistance patterns. The isolates were primarily from nonoutbreak situations and included 114 USA300 and 100 non-USA300 isolates (Table (Table1).1). Ninety-two S. aureus USA300 isolates were resistant to oxacillin and contained the SCCmec type IV element. Of these, 82 contained SCCmec IVa (63 had PFGE type USA300-0114), while 7, 1, and 2 contained SCCmec IVb, IVc, and type IV elements that could not be classified as either a, b, or c (SCCmec IVmisc), respectively. Twenty-two USA300 isolates did not contain SCCmec and were phenotypically methicillin (oxacillin)-susceptible S. aureus. Of these, nine had SmaI pulsed-field patterns similar to that of USA300-0114, but with either a 30-kb or a 60-kb reduction in the size of the SmaI fragment encoding mecA. The non-USA300 MRSA isolates included representatives of PFGE patterns USA100 through USA1100; the common European community-associated ST80 strain; and isolates exhibiting the Iberian clone PFGE pattern (i.e., CC8, like USA300, but ST247 rather than ST8) (4).

TABLE 1.
S. aureus isolates tested for arcA

Antimicrobial susceptibility profiles were determined by broth microdilution using panels prepared at the CDC as described previously (3). PFGE analysis (7), PCR detection of mecA (6) and the arcA region of ACME (5), and SCCmec typing (17) were also performed as described previously. For select ACME-positive USA300 MSSA isolates, the junction between ACME and orfX was amplified using the orfX-specific PCR primer Xsau325 (5′-GGATCAAACGGCCTGCACA-3′) (9) and a reverse primer within ACME, AcmeR (5′-CCTCCTTCACTTAGCACTG-3′). PCR conditions were 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with a final extension at 72°C for 5 min. Products were sequenced using an ABI Prism 3100 Avant genetic analyzer (Applied Biosystems, Foster City, CA).

All 82 CC8:ST8 USA300 MRSA isolates harboring SCCmec IVa recovered from 2001 to 2005 were positive for the ACME arcA gene by PCR. These isolates were obtained from diverse geographic locations in the United States (Table (Table1).1). However, arcA was not detected in CC8:ST8 USA300 isolates containing SCCmec type IVb, IVc, or IVmisc (Table (Table1).1). Three (7.0%) of 43 CC5:ST5 USA100 MRSA strains (42 from Nebraska, isolated in 2004 to 2005, and 1 from Ohio, isolated in 2000) were positive for arcA, as were 3 additional recent (2005) USA100 isolates from California. The remaining MRSA isolates belonging to other CCs were arcA negative. The majority of the USA300 MSSA isolates were arcA negative and SCCmec negative and demonstrated a SmaI fragment approximately 57 kb smaller than the corresponding fragment in S. aureus USA300-0114 (Fig. (Fig.1,1, lanes 1 and 3). Thus, SCCmec and ACME integrate into the same SmaI fragment (5). However, 2 of 22 USA300 MSSA isolates (from California and Nevada) were arcA positive with a single SmaI fragment ~30 kb smaller than the corresponding mecA-containing fragment in USA300-0114 (Fig. (Fig.1,1, lane 2), suggesting at least some loss of SCCmec. DNA sequence analysis confirmed the complete absence of SCCmec from identical 333-bp PCR products generated from the two MSSA strains using ACME- and orfX-specific primers (Fig. (Fig.2).2). Compared to the published USA300 genome, the sequences contained only the direct repeat found at the ACME-SCCmec junction (DRACME-R) and lacked the highly related repeat (DRSCCmec-R) associated with the SCCmec-orfX junction (5).

FIG. 1.
SmaI-digested chromosomal DNA from S. aureus isolates USA300-0114 (mecA positive, arcA positive) (lane 1), USA300-0283 (mecA negative, arcA positive) (lane 2), and USA300-0020 (mecA negative, arcA negative) (lane 3), analyzed by BioNumerics, version 4.1. ...
FIG. 2.
Diagram of a portion of the published USA300 chromosomal sequence (GenBank accession number CP000255) showing ACME, SCCmec, and orfX (5). The numbers above ...

Our results confirm that ACME is associated with USA300 isolates but is currently limited to those containing SCCmec type IVa, a category that includes the widely disseminated strain USA300-0114 (10, 20), and a limited number of USA100 isolates. Other USA300 PFGE subtypes containing SCCmec IVb, SCCmec IVc, or other variants did not appear to harbor ACME. ACME was not detected in representative isolates of other successful community-associated MRSA strains, including USA400 (1, 16), USA1000 (18), USA1100 (12, 13), and European ST80 isolates (8). The absence of ACME in these community-associated strains does not negate its potential role as a virulence factor but suggests that other virulence factors are present in these isolates. We identified arcA in six isolates of USA100, the multiply resistant health care-associated lineage of MRSA (14), which continues to cause a significant number of infections in U.S. hospitals (11). The mechanisms underlying the apparently limited association of ACME with specific SCCmec types (i.e., IVa and II) and MLST CCs (thus far CC8 and CC5) are unknown. ACME may well be associated with additional MLST CCs over time.

USA300 isolates harboring SCCmec IVa represent approximately 90% of the 988 USA300 isolates in the CDC S. aureus strain collection. USA300-0114 represents 66.3% of USA300 SCCmec IVa isolates, while subtypes USA300-0047 and USA300-0247 represent 10.5% and 4.8% of USA300 isolates, respectively. Isolates carrying SCCmec IVb (e.g., USA300-0045 and USA300-0120) represent only 8.2% of all 988 USA300 isolates, while SCCmec IVc and others are less than 2% of USA300 isolates. Thus, ACME is present in the majority of USA300 isolates collected by the CDC since 2000.

The location of ACME adjacent to SCCmec at orfX on the S. aureus chromosome suggests a role of SCCmec recombinase (ccr) genes in ACME integration (5). However, no SCCmec sequences were identified in MSSA arcA-positive USA300 isolates. This is likely explained by the precise deletion of SCCmec, including deletion of the direct repeat (DRSCCmec-R) at the SCCmec-orfX junction but leaving the direct repeat at the ACME-SCCmec junction (DRACME-R). However, integration of ACME into an MSSA strain cannot be completely ruled out.

Acknowledgments

The findings and conclusions in this publication are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 April 2007.

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