LAUSR

Sir, MSSA strains can become methicillin resistant through the acquisition of mecA or mecC, which are usually located on a mobile genetic element termed staphylococcal cassette chromosome mec (SCCmec). In addition, a transferable plasmid-borne mecB gene, which also accounts for methicillin resistance, has recently been identified in Staphylococcus aureus. The SCCmec is composed of two major parts: the mec gene complex, containing the mec genes, and the ccr gene complex, consisting of site-specific recombinase (ccr) genes responsible for the integration and excision of the element into and from the staphylococcal chromosome. To date, at least 13 major types and numerous subtypes have been identified in different staphylococcal species. The aim of this study was to gain insight into the structures of SCCmec elements from two closely related ST9 MRSA isolates (QD-CD9 and NX-T55), one of which lacks ccr genes. The MRSA isolates, both of which belonged to clonal complex 9 and spa type t899, were obtained from nasal swabs collected from healthy pigs during our previous surveillance study. Isolate NXT55 contained mecA, but was negative for all known ccr genes. We therefore proposed that a novel SCCmec element may exist in NX-T55. Genomic DNA from each of the two isolates was sequenced using a PacBio RSII sequencer. Genome assemblies were performed using HGAP and Quiver in SMRTAnalysis using the HGAP3 protocol, and were corrected using Pilon. Genome analysis revealed that the two MRSA isolates contained different, but related, SCCmec elements. The SCCmec element of isolate QD-CD9 was similar to the recently described SCCmec XII element found in MRSA strain BA01611 (GenBank accession number NZ_CP019945.1). The SCCmec element of isolate BA01611 harboured ccrC2 and was flanked by a pseudo-SCC element carrying a truncated ccrA1 gene. Compared with the SCCmec XII element of isolate BA01611, the type C2 mec gene complex in isolate QD-CD9 was in the opposite orientation. In addition, the QD-CD9 SCCmec element was disrupted by core S. aureus chromosomal DNA, splitting the element into two segments of 41.6 and 12.2 kb. The 41.6 kb segment contained the key structures of the type XII SCCmec element (ccr and mec gene complexes) as well as the pseudo-SCC element. The 12.2 kb segment contained the J3 region of SCCmec XII along with two additional IS431 elements and an IS256 element (Figure 1). A novel pseudo-SCCmec (/SCCmecT55) element was identified in isolate NX-T55. The 29.2 kb /SCCmecT55 contained a relic of SCCmec XII with the class C2 mec gene complex, but was negative for the ccr genes. Compared with the QD-CD9 SCCmec, a 19.3 kb segment was absent from /SCCmecT55. This segment encompassed the region from the truncated ccrA1 gene to immediately upstream of the first IS431 element and included the entire ccr gene complex. A 3.2 kb fragment containing the type III restriction-modification system methylation subunit bracketed by two copies of IS431 in the same orientation was also lost in isolate NX-T55 and was replaced by a single copy of IS431 (Figure 1). The two SCCmec elements identified in the current study are completely different from the type III and IV elements previously identified in pig-associated MRSA isolates from China. The mechanism involved in the emergence and evolution of SCCmec in MRSA remains unclear, but the ccr genes appear to be involved in the horizontal transfer of the SCCmec element. There is also evidence suggesting that the insertion sequence IS431, which is ubiquitous in all SCCmec regions, may play a role in the movement and stability of mecA. All variable regions within the SCCmec elements in the three isolates compared in this study, except the segments containing the ccr gene complex, appeared to have an abundance of IS431 elements. Interestingly, a high frequency and diversity of ccr genes was detected in methicillin-susceptible Staphylococcus sciuri isolates, which may provide the components for SCCmec integration and assembly. In addition, /SCCmec elements have occasionally been found in other staphylococci. While it is not known how the ccr and mec genes integrate into SCCmec, the fact that the mec and the ccr gene complexes are present on an individual genetic element in staphylococci indicates that other unknown mechanisms may also be involved. Our findings may indicate that the /SCCmecT55 element found in isolate NX-T55 represents either a deletion derivative of a formerly complete SCCmec XII element or a preliminary version into which a ccr gene complex was inserted during the formation of SCCmec XII.