Molecular and Cellular Probes 22 (2008) 1–13 Development and validation of microarray-based assay for epidemiological study of MRSA Junnosuke Otsuka a,b , Yasumitsu Kondoh a , Tomoyuki Amemiya a , Akio Kitamura c , Teruyo Ito c , Satoshi Baba c , Longzhu Cui c , Keiichi Hiramatsu c , Tomoko Tashiro b , Hideo Tashiro a,Ã a Probing Technology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan b Department of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Sagamihara, Kanagawa 229-8558, Japan c Department of Bacteriology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Received 30 January 2007; accepted 18 May 2007 Available online 5 June 2007 Abstract We have developed a microarray-based assay for the genotyping of Staphylococcus aureus strains. A DNA microarray consisting of 221 genes with 390 oligonucleotide probes was designed to identify characteristic genes or gene alleles of S. aureus. The 221 genes were chosen on the basis of the following criteria: (i) genes used as control for the microarray system, (ii) virulence genes, (iii) resistance genes and their regulators, and (iv) genes constituting genomic islands, e.g., SCCmec. The microarray system was established by determining the method to prepare targets by random-primer labeling with chromosomal DNA and the conditions for hybridization. We verified the system by using DNAs of seven strains, the genome of which has been fully sequenced. Furthermore, the presence of 32 genes and the types of SCCmec elements and coagulase genes carried by another 27 strains were examined and compared with the results of PCR. As a result, the presence or absence of 182 genes out of the 221 genes was verified. Our data showed the usefulness of the oligonucleotide microarray based assay in identifying important marker sets, such as toxin genes, resistance genes, SCCmec elements, and coagulase genes, for the molecular epidemiology of S. aureus. r 2007 Elsevier Ltd. All rights reserved. Keywords: MRSA; Epidemiological study; Oigonucleotide microarray 1. Introduction Staphylococcus aureus is the most notorious staphylo- coccal species because of its frequent and highly versatile pathogenicity in humans and animals. Enterotoxins, exfoliative toxins, and toxic shock syndrome toxins are known to be responsible for various pathologies [1]. The coordinated action of several genes that are expressed under the control of several regulatory systems also causes clinical symptoms [2,3]. S. aureus is one of the most common causes of hospital infection. This has become a great matter of concern because more than half of S. aureus strains in hospitals are methicillin-resistant, or MRSAs, which are resistant to most of the antibiotics used in hospitals [4,5]. In particular, MRSA strains that exhibit decreased susceptibility to glycopeptides [6–8] have ex- tremely limited our choice of antibiotics for the treatment of hospital-acquired S. aureus infections. Although the molecular events responsible for human pathogenesis are not understood completely, many genes are presumed to be involved, and their allelic variations among strains are considered to influence the pathogenic potential and the degree of drug resistance of each MRSA strain [9]. Analysis of variance of the gene repertoire among strains, therefore, would greatly enrich our knowledge of the mechanism of S. aureus pathogenicity. S. aureus is characterized con- ventionally by serological, microscopic, biochemical, phy- siological, and selective culture plating methods [10–14]. However, these phenotypic methods have low resolution. ARTICLE IN PRESS www.elsevier.com/locate/ymcpr 0890-8508/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.05.007 Ã Corresponding author. Tel.: +81 48 467 9303; fax: +81 48 467 9300. E-mail address: htashiro@riken.jp (H. Tashiro).