Variations in Epidemic Methicillin-Resistant Variations in Epidemic Methicillin-Resistant Staphylococcus aureus- Staphylococcus aureus- 16 (EMRSA-16) 16 (EMRSA-16) Stefan Monecke (1), Brigitte Berger-Bächi (2), Geoffrey Coombs (3,6), Anne Holmes (4), Ian Kay (3), Angela Kearns (4), Hans-Jörg Linde (5), Frances O´Brien (6), Peter Slickers (7), Ralf Ehricht (7) (1) Institute for Medical Microbiology and Hygiene, Technical University of Dresden, Germany, (2) Institute of Medical Microbiology, University of Zurich, Switzerland, (3) Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine Western Australia - Royal Perth Hospital, Australia, (4) Staphylococcus Reference Unit, Centre for Infections, Health Protection Agency, London, UK, (5) Institute for Medical Microbiology and Hygiene, University of Regensburg, Germany, (6) Gram-positive Bacteria Typing and Research Unit, School of Biomedical Sciences, Curtin University of Technology, Perth, Australia, (7) CLONDIAG chip technologies GmbH, Jena, Germany Staphylococcus aureus of MLST 30 and 36 appear to be especially successful in evolutionary terms. This complex comprises several epidemic strains such as the ancient pandemic phage group 80/81, the majority of TSS-positive isolates, the community-associated “Southwest Pacific PVL-MRSA”, and the healthcare-associated EMRSA-16. The latter is a SCC mec type II MRSA strain which is widespread and represents one of the predominant MRSA strains in the United Kingdom. Two reference strains (Sanger MRSA252 and ATCC43300) together with seven clinical isolates were characterised using microarray hybridisation, MLST, and spa-typing. They belonged to MLST type 36 (with the exception of ATCC43300 which was MLST type 39) and spa- types 7, 12, 18, or 419. Generally, isolates were similar to the sequenced strain Sanger MRSA 252 in regard to the presence of mecA, ermA, enterotoxin genes G, M, N, O, and U, haemolysins, lukS/F-hlg , and set-genes. Some notable variations were found regarding resistance and toxin genes. Four clinical isolates carried a second macrolide resistance determinant, ermC. These isolates lacked aadD, indicating a deletion/mutation in the SCCmec type II cassette, but harboured aacA-aphD which confers resistance to gentamicin. ATCC43300 was unique in carrying both, aadD and aacA-aphD. Four isolates were positive for mupR, which is associated with high-level resistance to mupirocin. The sequenced strain Sanger MRSA252 was atypical as it was the only EMRSA-16 strain tested which did not contain the gene for the toxic shock syndrome toxin (tst1). In contrast to all other strains, ATCC43300 lacked enterotoxin genes seA and seI , but seC and seL were detected. These data emphasise the high plasticity of the S. aureus genome and highlight clinically relevant changes even within a recently emerged outbreak strain. Introduction: Staphylococcus aureus of the related MLS types 30 and 36 appear to be especially successful in evolutionary terms. This complex comprises several clinically important epidemic strains. The oldest yet described ancestor form is represented by the PVL-positive, drug-susceptible strain ATCC 25923. Later on, the beta-lactamase producing phage group 80/81 and the community-associated “Southwest Pacific PVL-MRSA” (aka. WSPP, ”West Samoan Phage Pattern Clone”) emerged and spread worldwide. A majority of TSS-positive isolates also belongs to this complex. Another branch of this lineage is the healthcare-associated strain EMRSA-16. In contrast to WSPP, it is a SCCmec type II MRSA strain. It is is widespread and represents one of the predominant MRSA strains in the United Kingdom. Material & Methods: Array preparation: 144 genes (or distinct allelic variants thereof) were selected which represent genus- and species-specific controls, all relevant resistance genes, virulence markers / exotoxins, set -locus genes, and allelic variants of the agr regulatory locus. Probe (as well as primer) sequences were derived from consensus regions of all published target gene sequences. 3´-aminomodified oligonucleotides were purchased (Metabion) and spotted on arrays which were inserted into ArrayTube TM reaction vials (Clondiag). Strains and isolates : Two reference strains, sequenced strain Sanger MRSA252 and ATCC43300, as well as four clinical isolates were tested. Strains were cultured on Columbia blood broth. Staphylococcal cells were lysed in two steps using a buffer containing lysostaphin and lysozym and a commercial lysis buffer (QIAGEN) plus proteinase K [1, 2]. DNA was prepared using the QIAGEN device EZ1. Amplification and Hybridisation: For every target, one specific primer was developed, and a mixture of these primers was used for a multiplex primer elongation reaction incorporating Biotin-16-dUTP into the amplicons. The labelled sample was hybridised against the DNA-array and horseradish peroxidase coupled streptavidin was added. Then, the ArrayTube was placed into a reading device (ATR 01 TM , Clondiag). TMB Peroxidase Substrate was added for staining. Scans were recorded and analysed. The procedure has been described elsewhere in detail [1, 2]. Other typing methods: Additionally, MLST , spa- and SCCmec types were determined using previously published methods. . Results and Discussion: Array hybridisations made it possible to screen isolates of a single epidemic strain or clonal group for changes such as the acquisition of resistance determinants. This approach was applied to the ST36-MRSA II, also known as EMRSA-16. All isolates contained mecA , the enterotoxin gene cluster ( egc) comprising enterotoxin genes G, I, M, N, O, U, and Y as well as a characteristic set of allelic variants of set-genes. However, within the EMRSA-16 complex, some notable variations were observed. Concerning antibiotic resistance determinants, the reference strains as well as two clinical isolates contained ermA and aadD. This is consistent to the presence of a SCCmecII element. Four isolates lacked aadD. The reason for the truncation of the SCCmec II element is not clear. An explanation could be that a loss of parts of the SCCmecII element reduced possible fitness costs of its presence (such as slower growth [3]). Five strains (including ATCC43300) harboured another aminoglycoside resistance gene, aacA-aphD. It confers resistance to gentamicin and tobramycin, effectively more than just compensating for the loss of aadD. Four isolates carried a second macrolide-/lincosamide resistance determinant, ermC, and three isolates were positive for dfrA (trimethoprim resistance). Four isolates also contained mupR. The acquisition of that gene is clinically relevant since it confers resistance to mupirocin which is used as nasal ointment for decolonising patients with MRSA. Carriage of virulence factors was also affected by inter-strain variation. The sequenced strain Sanger MRSA252 did not contain tst1 which was found in all clinical isolates. ATCC43300 lacked enterotoxin genes seA and seI. As these strains are laboratory strains, it could be possible that these virulence factors were lost in the course of culture passages under in vitro conditions, i.e., in absence of a host defence. ATCC43300 was positive for enterotoxin genes seC and seL which did not occur in other EMRSA-16 strains. These variations within different isolates belonging to a single outbreak strain emphasise the high plasticity of the S. aureus genome. The clinical relevance of these changes affecting both, virulence and drug resistance, warrants further surveillance. Acknowledgments: This work was supported partially by a grant from the Technical University of Dresden. The authors want to thank Antje Ruppelt, Susann Kolewa, Elke Müller, Ute Schmitt, and Kathrin Weitze for technical assistance. We are indebted to the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for supplying reference strains. Literature: 1. Monecke S, Ehricht R. Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays. Clin Microbiol Infect 2005; 11: 825-833. 2. Monecke S, Slickers P, Hotzel H et al. Microarray-based characterisation of a Panton-Valentine leukocidin-positive community-acquired strain of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 2006; 12: 718-728. 3. Ender M, McCallum N, Adhikari R et al. Fitness Cost of SCCmec and Methicillin Resistance Levels in Staphylococcus aureus. Antimicrob. Agents Chemother. 2004; 48: 2295-2297. Figure 1: Sequenced strain Sanger MRSA252 as well as two clinical EMRSA-16 isolates on the DNA-arrays. Differences in the carriage of several resistance genes (blue and lilac squares) and tst1 (gene encoding toxic shock toxin, red squares) are visible. Figure 2: The presence of relevant antibiotic resistance determinants and virulence genes in study strains as well as MLST and spa-typing data. View publication stats View publication stats