A Clonal Complex 12 Methicillin-Resistant Staphylococcus aureus Strain, West Australian MRSA-59, Harbors a Novel Pseudo-SCCmec Element

Methicillin-resistant Staphylococcus aureus (MRSA) isolates harbor mecA or mecC genes, which encode modified penicillin-binding proteins that confer resistance toward most beta-lactam compounds (1–3). The mec genes are located on staphylococcal cassette chromosome mec (SCCmec) elements that facilitate horizontal gene transfer in staphylococci. They contain a mec complex which, in addition to mecA or mecC, consists of various combinations of regulatory genes and insertion sequences (4). Furthermore, they harbor a recombinase gene (ccr) complex and the so-called J-regions (“joining” or “junkyard” regions). J-regions include various genes, including additional resistance or virulence determinants. Based on the combination of different mec and ccr complexes, 11 types of SCCmec elements have been described (4) (http://www.sccmec.org/Pages/SCC_TypesEN.html), and subtypes are differentiated based on variations within the J-regions. Truncated SCCmec elements lacking ccr recombinase genes are known as pseudo-SCCmec elements (5). In addition to SCCmec elements, a variety of different SCC elements have been described that lack mecA but harbor other markers, such as fusC, which encodes fusidic acid resistance (5, 6).

In Western Australia (WA), a state-wide MRSA management policy has prevented the transmission of health care-associated MRSA in acute care hospitals (7). However, community-associated MRSA is endemic in the region (8). To distinguish between health care- and community-associated MRSA, all isolates from WA are referred to the Australian Collaborating Center for Enterococcus and Staphylococcus Species in Perth.

One of the isolates, 07-16590, designated Western Australian MRSA-59 (WA MRSA-59), was found to carry a novel SCCmec element. It was isolated in 2007 from sputum of a 56-year-old female patient treated for a chronic pulmonary Mycobacterium intracellulare infection. Isolate 07-16590 was assigned to multilocus sequence type 12 and Ridom spa type t160. An additional isolate was recovered during a follow-up examination of the index patient in 2008. Two other WA MRSA-59 isolates were recovered in 2008 and 2013, but there were no known epidemiological links.

DNA microarray analyses were performed on these four isolates as previously described (9). In short, S. aureus was grown on blood agar and enzymatically lysed. Purified DNA was subjected to a multiplex linear primer elongation that was used for amplification and labeling of virulence- and resistance-associated genes, various typing markers, and species-specific controls (9). Resulting biotin-labeled amplicons were hybridized to arrays with spotted specific oligonucleotides. Hybridizations were visualized by use of a streptavidin-horseradish peroxidase conjugate that triggered a local dye precipitation. Array images were automatically analyzed with regard to the absence or presence of the target genes as well as to strain/clonal complex (CC) assignment.

Like all CC12 isolates, WA MRSA-59 belongs to agr group II and capsule type 8. Isolates harbor the enterotoxin homologue open reading frame (ORF) CM14 and cna gene (for collagen-binding adhesin). The sak gene (for staphylokinase), scn (for staphylococcal complement inhibitor), and the enterotoxin genes sea-N315 (sep) were detected; according to the sequence analysis, they were localized on a hemolysin beta-integrating phage. The enterotoxin B gene (seb) was variably present. The isolates lacked tst-1 (toxic shock syndrome toxin gene) and the genes encoding exfoliative toxins and the Panton-Valentine leukocidin. The carriage of blaZ (beta-lactamase) and of erm(C) (erythromycin/clindamycin resistance) genes was variable.

Sequencing of isolate 07-16590 was undertaken by Geneservice Source BioScience PLC (Nottingham, United Kingdom) and by GATC Biotech (Constance, Germany) using the Illumina genome analyzer system. Reads were assembled into contigs by using the Velvet de novo genome assembler. Contigs were analyzed using various in-house scripts. Illumina sequencing left a gap of approximately 1,000 bp that bisected the SCC sequence, which was most likely caused by difficulties for the assembler in coping with multicopy sequences, such as IS431. The gap was closed by conventional sequencing using the primers listed in Table 1. The relevant region was amplified with primers mecI_02 and copA2_02, resulting in an approximately 1,500-bp amplicon. After initial denaturation (60 s at 96°C), 35 cycles of denaturation (15 s at 96°C), annealing (60 s at 50°C), and elongation (90 s at 72°C) were used. The PCR was finished with another elongation step (60 s at 72°C). DNA sequencing was carried out using the primers given in Table 1, the BigDye Terminator v1.1 cycle sequencing kit, and an ABI Prism 3130 genetic analyzer (both from Applied Biosystems, Darmstadt, Germany).

The SCCmec element of WA MRSA-59 isolate 07-16590 (Fig. 1; Table 2) consisted of dcs (downstream constant segment, locus 1), Q9XB68-dcs (truncated putative protein), tnp IS431 (transposase for IS431), mvaS-SCC (truncated 3-hydroxy-3-methylglutaryl coenzyme [CoA] synthase), Q5HJW6 (putative protein), dru (direct repeat units, type dt8b), ugpQ (glycerophosphoryl diester phosphodiesterase), ydeM (putative dehydratase), txbi mecA (bidirectional rho-independent terminator of mecA), mecA (modified penicillin-binding protein 2a [PBP2a]), mecR1 (signal transducer protein MecR1), mecI (methicillin resistance regulatory protein), txbi mecI (bidirectional rho-independent terminator of mecI, with an insertion of an inverted repeat for IS431, IR-IS431, at its downstream end), tnp IS431, copA2 (copper-exporting ATPase), mco (multicopper oxidase), ydhK (putative lipoprotein; GenBank accession number A8YZ03), arsC-arsB-arsR (arsenic resistance gene cluster), ORF PT43 (putative protein associated with arsenic resistance operon from SCCmec IX of S. aureus JCSC6943; GenBank accession number AB505628.1), and per-2 (plasmidic permease).

SCCmec-associated recombinase genes ccrA-ccrB and ccrC, xylR (mecR2) (homologue of xylose repressor), and the gene encoding PSM-mec (SCC-associated phenol-soluble modulin) were not detected.

Previously characterized SCCmec elements with mec gene complex A (SCCmec types II, III, and VIII) harbor genes mecA, mecI, mecR1, PSM-mec, and xylR (mecR2). Based on the observations described herein, we suggest that a second subtype of the mec complex A should be recognized. It is characterized by absence of PSM-mec and xylR (mecR2) as well as by insertion of IR-IS431 into the downstream end of txbi mecI. A search of the WGS section of GenBank uncovered that the mec complex identified in isolate 07-16590 is also present in Staphylococcus hominis M0480 (KK013382.1/JCGQ), S. hominis ZBW5 (AKGC), and Staphylococcus epidermidis VCU120 (AHLC), suggesting that horizontal gene transfer between different species might have occurred.

A part of the SCCmec element of S. epidermidis VCU120 is very similar to that of WA MRSA-59, as it harbors the same mec gene complex and also copper and arsenic resistance operons in a comparable configuration. However, VCU120 also harbors a ccrB4 recombinase gene and the ACME 1 element (opp3B-opp3C, arcA-arcB-arcC-arcD). Since the SCC-associated genes in VCU120 are spread across several contigs, their relative locations have not been elucidated. The other two sequences differ with respect to the presence of czrC (copA; zinc/copper resistance), of multiple ccr genes, and (in M0480 only) of fusC accompanied by tirS.

The pseudo-SCCmec element of 07-16590 and other SCCmec elements harbor arsenic and copper resistance factors. The evolutionary benefit of heavy metal resistance operons in staphylococci warrants further investigation. These operons are very common, not only in their core genomes but also in SCC elements, and many SCCmec elements comprise multiple, and redundant, heavy metal resistance genes. Selective pressures favoring acquisition and maintenance of heavy metal resistance genes may include environmental exposure, past medical use of heavy metals, the use of heavy metals as growth promoters in veterinary medicine, or a coselection when localized on mobile genetic elements together with genes encoding antibiotic resistance.

In conclusion, we have described a novel pseudo-SCCmec element in S. aureus and we suggest that mec complex A should be split into two subtypes, based on the mutually exclusive presence of (i) PSM-mec and xylR (mecR2) or (ii) the insertion of IR-IS431 at the downstream end of txbi mecI.

We thank Hui-Leen Tan (Perth), Antje Ruppelt (Dresden), and Ines Engelmann, Elke Müller, and Annett Reissig (Jena) for excellent technical help and assistance.