In recent years, methicillin-resistant Staphylococcus aureus (MRSA) have become a truly global challenge. In addition to the long-known healthcare-associated clones, novel strains have also emerged outside of the hospital settings, in the community as well as in livestock. The emergence and spread of virulent clones expressing Panton-Valentine leukocidin (PVL) is an additional cause for concern. In order to provide an overview of pandemic, epidemic and sporadic strains, more than 3,000 clinical and veterinary isolates of MRSA mainly from Germany, the United Kingdom, Ireland, France, Malta, Abu Dhabi, Hong Kong, Australia, Trinidad & Tobago as well as some reference strains from the United States have been genotyped by DNA microarray analysis. This technique allowed the assignment of the MRSA isolates to 34 distinct lineages which can be clearly defined based on non-mobile genes. The results were in accordance with data from multilocus sequence typing. More than 100 different strains were distinguished based on affiliation to these lineages, SCCmec type and the presence or absence of PVL. These strains are described here mainly with regard to clinically relevant antimicrobial resistance- and virulence-associated markers, but also in relation to epidemiology and geographic distribution. The findings of the study show a high level of biodiversity among MRSA, especially among strains harbouring SCCmec IV and V elements. The data also indicate a high rate of genetic recombination in MRSA involving SCC elements, bacteriophages or other mobile genetic elements and large-scale chromosomal replacements.

A diagnostic microarray was used to characterise a collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates from hospitals in the German region of Eastern Saxony. The most abundant epidemic MRSA (EMRSA) strains were ST5-MRSA II (Rhine-Hesse EMRSA, EMRSA-3), CC5/ST228-MRSA I (South German EMRSA), ST22-MRSA IV (Barnim EMRSA, EMRSA-15) and ST45-MRSA IV (Berlin EMRSA). Other strains were found only as sporadic isolates or in minor outbreaks. These strains included ST1-MRSA IV, ST8-MRSA IV (Hannover EMRSA and others), clonal group 5 strains carrying SCCmec type IV elements (Paediatric clone), ST45-MRSA V, CC8/ST239-MRSA III and ST398-MRSA V. Panton-Valentine leukocidin-positive MRSA isolates were still very rare. The predominant strain was ST80-MRSA IV, although increasing numbers of different strains have recently been detected (ST8-MRSA IV, ST30-MRSA IV and ST59-MRSA V). For more common MRSA strains, it was possible to detect variants that differed mainly in the carriage of additional resistance determinants and certain virulence-associated genes. Detection of such variants can sometimes allow epidemic strains to be resolved beyond spa types to a hospital-specific level, which is of significant value for epidemiological purposes.

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.

Worldwide, methicillin-resistant Staphylococcus aureus (MRSA) pose an increased risk for healthcare- and community-associated infections. Since the first report of MRSA in England in 1961, several distinct clones or strains have emerged. Changes within the MRSA population of whole countries, small regions or of single hospitals have been observed with some clones replacing others. In this study, the clonal replacement of MRSA isolates in a South-eastern German tertiary care hospital between 2000 and 2010 is described based on microarray analyses of 778 isolates and at least 50 MRSA per year. Within these eleven years, four common epidemic strains, CC22-MRSA-IV, CC45-MRSA-IV, CC5/ST228-MRSA-I (including a variant with a truncated SCCmec element) and CC5-MRSA-II were identified. The PVL-negative CC22-MRSA-IV strain (Barnim Epidemic Strain, UK-EMRSA-15) was detected for the first time in 2001 and its abundance increased since then to 58.6% in 2010. CC5-MRSA-II increased from 2% (2000) to about 30% (2003), and since then it fluctuates between 23 and 37% of isolates. CC5/ST228-MRSA-I decreased from about the half of tested isolates (2000) to 2.3% (2010). A similar trend was observed for CC45-MRSA-IV, which decreased drastically down to 3.4% in 2010 after reaching a maximum of 62.0% in 2002. Seventeen other PVL-negative MRSA strains were identified sporadically with no significant trend being observed. Seven PVL-positive MRSA strains were found, but they remained rare during the study period accounting together for 2.7% of isolates.

ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramírez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.