Abstract
SCCmec elements are very important mobile genetic elements in Staphylococci that carry beta-lactam resistance genes mecA/mecC, recombinase genes and a variety of accessory genes. Twelve main types and a couple of variants have yet been described. In addition, there are also other SCC elements harbouring other markers. In order to subtype strains of methicillin-resistant S. aureus (MRSA) based on variations within their SCCmec elements, 86 markers were selected from published SCC sequences for an assay based on multiplexed primer extension reactions followed by hybridisation to the specific probes. These included mecA/mecC, fusC, regulatory genes, recombinase genes, genes from ACME and heavy metal resistance loci as well as several genes of unknown function. Hybridisation patterns for published genome or SCC sequences were theoretically predicted. For validation of the microarray based assay and for stringent hybridisation protocol optimization, real hybridization experiments with fully sequenced reference strains were performed modifying protocols until yielded the results were in concordance to the theoretical predictions. Subsequently, 226 clinical isolates from two hospitals in the city of Dresden, Germany, were characterised in detail. Beside previously described types and subtypes, a wide variety of additional SCC types or subtypes and pseudoSCC elements were observed as well as numerous composite elements. Within the study collection, 61 different such elements have been identified. Since hybridisation cannot recognise the localisation of target genes, gene duplications or inversions, this is a rather conservative estimate. Interestingly, some widespread epidemic strains engulf distinct variants with different SCCmec subtypes. Notable examples are ST239-MRSA-III, CC5-, CC22-, CC30-, and CC45-MRSA-IV or CC398-MRSA-V. Conversely, identical SCC elements were observed in different strains with SCCmec IVa being spread among the highest number of Clonal Complexes. The proposed microarray can help to distinguish isolates that appear similar or identical by other typing methods and it can be used as high-throughput screening tool for the detection of putative new SCC types or variants that warrant further investigation and sequencing. The high degree of diversity of SCC elements even within so-called strains could be helpful for epidemiological typing. It also raises the question on scale and speed of the evolution of SCC elements.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens in hospitals and the community
Twelve different types of SCCmec elements have so far been described ([4,5,6,7,8]; http://www.sccmec.org/Pages/SCC_TypesEN.html). Their nomenclature relies on the identity of the mec complex, i.e., the immediate surroundings of mecA, including its regulatory genes, and on the identity of the recombinase gene complex [6]
Probes including those that were used for clonal complex determination as well as detection of toxin genes and resistance genes have been previously discussed, and these probe and primer sequences were provided elsewhere [11,12]
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens in hospitals and the community. In 2011, a second gene, mecC has been discovered that causes methicillin/beta-lactamresistance [2,3] Both genes are situated on large, potentially mobile genetic elements, so-called SCCmec elements (staphylococcal cassette chromosome mec). These elements harbour regulatory genes, recombinase genes and a variety of accessory genes. Twelve different types of SCCmec elements have so far been described ([4,5,6,7,8]; http://www.sccmec.org/Pages/SCC_ TypesEN.html) Their nomenclature relies on the identity of the mec complex, i.e., the immediate surroundings of mecA, including its regulatory genes, and on the identity of the recombinase gene (ccr) complex [6]. Due to variations within the J-regions some SCCmec types can further be differentiated into subtypes
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