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Molecular and Cellular Probes 22 (2008) 113 www.elsevier.com/locate/ymcpr

Development and validation of microarray-based assay for epidemiological study of MRSA

Junnosuke Otsukaa,b, Yasumitsu Kondoha, Tomoyuki Amemiyaa, Akio Kitamurac, Teruyo Itoc, Satoshi Babac, Longzhu Cuic, Keiichi Hiramatsuc, Tomoko Tashirob, Hideo Tashiroa,
b a Probing Technology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 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 veried 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 veried. 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 staphylococcal 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,
Corresponding author. Tel.: +81 48 467 9303; fax: +81 48 467 9300.

E-mail address: htashiro@riken.jp (H. Tashiro). 0890-8508/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2007.05.007

which are resistant to most of the antibiotics used in hospitals [4,5]. In particular, MRSA strains that exhibit decreased susceptibility to glycopeptides [68] have extremely 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 inuence 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 conventionally by serological, microscopic, biochemical, physiological, and selective culture plating methods [1014]. However, these phenotypic methods have low resolution.

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Even S. aureus strains that are deemed identical by such phenotypic methods may actually be different when grown on various natural substrates and laboratory media [15]. In particular, serological methods cannot identify a large group of related proteins having signicant antigenic similarities, such as enterotoxins. In recent years, a variety of molecular genotyping techniques have emerged to analyze S. aureus gene diversity in a rapid and precise manner [1619]. Pulse eld gel electrophoresis (PFGE) is popularly used as a tool for molecular epidemiology [20]. However, it presents only the relatedness of strains and does not convey any information on the content of the genome or the phenotypic potential of strains. In addition, its procedure is complex and time consuming. Multilocus sequence typing (MLST) was introduced as a staphylococcal typing method [21]. Sequences of the internal fragments of seven housekeeping genes are determined for each isolate, thereby dening the specic alleles for each locus. However, although MLST can classify strains into evolutionarily relevant groups, it does not provide any biologically or medically pertinent information. In view of the above situation, the microarray system is expected to be a powerful epidemiological tool because it can collectively detect thousands of genes or target DNA sequences on a single glass slide [22]. Although the microarray methods used in most studies have focused on the study of gene expression [23,24], they can be adopted for the DNA-based typing of specic pathogenic bacterial strains [2528]. Two oligonucleotide microarrays for S. aureus have been constructed [29,30]. StaphChip comprises 5427 probes covering approximately 90% of three S. aureus strain genomes [29], and is effective for whole-genome analysis. However, from a practical standpoint, probes should be made for selected genes because the synthesis of many probes is generally a very expensive task. The other microarray consists of 383 probes covering virulence genes [30]. However, the sensitivity and reliability of the oligonucleotide microarray have not been proven so far. More trials are needed to design a medically useful DNA microarray having sufciently high precision and reliability. Here, we have designed and tested a DNA microarray with gene-specic oligonucleotide probes to detect not only important genes but also the types of genomic islands carried by each MRSA strain. A set of probes were primarily designed to identify virulence genes. The microarray also contained probes designed for the detection of genomic islands. The most typical and important genomic island is SCCmec element, which carries drug resistance genes such as the methicillin resistance gene, mecA [3133]. The acquisition of methicillin resistance occurs via horizontal transfer of SCCmec from other staphylococcal strains. SCCmec has various types and subtypes and the detection of SCCmec type is expected to provide information relating to the origin of the MRSA strain. For example, SCCmec carried by health-care-associated MRSA

(H-MRSA) differs from that carried by communityacquired MRSA (C-MRSA) [34]. Therefore, SCCmec typing is essential for the characterization of clinical MRSA strains. Another important genomic island is pathogenicity island. High throughput gene sequencing of several S. aureus genomes has revealed the presence of large chromosomal regions carrying many virulence genes [35]. Representative pathogenic islands, nuSaa and nuSab, are found in practically all clinical strains, but in distinct allelic forms. The detection of the allelic types of the islands, therefore, would give us information on the evolutionary relationship of MRSA strains [36]. We report here the development of a new DNA microarray system that conveys both virulence and evolutionary information of MRSA strains. 2. Materials and methods 2.1. Bacterial strains Bacterial strains used in this experiment are listed in Table 1. We selected 32 S. aureus strains and two E. faecalis strains. 2.2. Total DNA preparation Total cellular DNA was extracted and puried with a MagPreps bacterial genomic DNA kit (Novagen, San Diego, CA), according to the manufacturers instructions. The extracted DNA was fragmented with a sonicator (VP-5S homogenizer, TAITEC, Japan) to an average size of 1000 bp. Concentration and purity of genomic DNA in the prepared samples were determined by measuring the absorbance at 260 and 280 nm with a spectrophotometer (Gene Spec V, Naka Instruments, Japan). 2.3. DNA labeling Fragmented DNA (1 mg) was labeled with chemically reactive nucleotide analog (aminoallyl-dUTP) by randomprimer labeling (BioPrimes plus array CGH genomic labeling system, Invitrogen, Carlsbad, CA) to generate aminoallyl-labeled DNA. Then, the aminoallyl-labeled DNA was coupled with Cy3-N-hydroxysuccinimide esters (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Cy3-labeled DNA was puried with Ultra PCR clean-up kit (ABgene, Rochester, NY) following the manufacturers instructions. The puried DNA was stored at 20 1C until use in hybridization. 2.4. Sequence design of specic oligonucleotide probes A total of 390 oligonucleotide probes were designed for 221 selected genes and DNA regions. The rst consideration was to choose regions specic to each gene and the

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J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 113 Table 1 Strains used in this study Strain ORFs used for veryfying microarray Coagulase serotype SCCmec type Source or references 3

Genome sequenced S. aureus strains N315 102 ORFs Mu50 108 ORFs MW2 96 ORFs NCTC8325 73 ORFs MRSA252 97 ORFs MSSA476 82 ORFs COL 81 ORFs

II II VII III IV VII III

IIa IIa IVa MSSA IIa MSSA I Not tested Not tested Iib Not tested Not tested Not tested Not tested I III IVa IVa IVb IVc IVd III V VI MSSA MSSA MSSA Not tested Not tested Not tested Not tested Not tested

Kuroda et al., 2001 [39] Kuroda et al., 2001 [39] Baba et al., 2002 [9] http://www.genome.ou.edu/staph.html Holden et al., 2004 [40] Holden et al., 2004 [40] Gill et al., 2005 [41] Provided by M. Sugai, Hiroshima Univ., Japan Provided by Y. Yoshizawa, Jikei Medical Univ., Japan Hisata et al., 2005 [6] Provided by K. Omoe, Iwate Univ., Japan Provided by K. Omoe, Iwate Univ., Japan Provided by K. Omoe, Iwate Univ., Japan Provided by J. Lee, Hervard Univ. USA Ito et al., 2001 [46] Ito et al., 2003 [32] Okuma et al., 2002 [47] Okuma et al., 2002 [47] Ma et al., 2002 [34] Ma et al., 2006 [48] Ma et al., 2006 [48] Ito et al., 2001 [46] Ito et al., 2003 [32] Provided by H. de Lencastre, Rockfeller, USA. Watanabe et al., 2005 [38] Watanabe et al., 2005 [38] Watanabe et al., 2005 [38] Provided by Y. Nakajima, Japan Provided by M. Matsuoka, Japan Provided by T. Ida, Meiji Seika Kaisha, Japan Provided by T. Ida, Meiji Seika Kaisha, Japan Provided by N. Noguchi, Tokyo phamacuetical Univ., Japan This study This study

S. aureus strains used for validation of specic genes or gene alleles TY114 Exfoliative toxin D (ETD) Not tested ZM Exfoliative toxin A (ETA) Not tested 030-1 196E FRI326 Fukuoka5 M NCTC10442 85/3907 JCSC1469 JCSC2167 8/6-3P 81/108 JCSC4469 85/2082 WIS HDE288 Ku JCSC4711 JCSC4712 NCTC8325(pI258) NCTC8325(pEP2104) MSC-3228 RN4220/pMF151 L20A Exfoliative toxin B (ETB) Staphylococcal enterotoxin (sea, sed, selj, selr) Staphylococcal enterotoxin (see, selq) Staphylococcal enterotoxin (selj, selr) Type-I capsule (capH1, capI1, capJ1) Type-I SCCmec, tetK Type-III SCCmec, tetK, ermB Type-IVa SCCmec, type VI coagulase Type-IVa SCCmec, type V coagulase Type-IVb SCCmec Type-IVc SCCmec Type-IVd SCCmec Type-III SCCmec, tetK Type-V SCCmec Type-VI SCCmec Type VIII coagulase Type IX coagulase Type X coagulase ermB msrA aac(60 )-aph(200 ), aphA-3 fosB Conjugative plasmid I Not tested Not tested Not tested Not tested IV IV VI V III IV Not tested Not tested I Not tested VIII IX X Not tested Not tested Not tested Not tested NT

E. faecalis strains used for validation 1658 VanA 1732 VanB

second was to avoid secondary structures with the use of Oligo 6.0 software (Molecular Biology Insights, Cascade, CO). The leading guidance was (i) 45 bp probe in length, (ii) 8278 1C in melting temperature (Tm), and (iii) o2.1 kcal/mol for hairpins. The specicity of the designed oligonucleotide probes was assessed with GENETYX-MAC software (Molecular Biology Insights, Cascade, CO) using the whole genome sequences of seven staphylococcal strains available in GenBank. We selected S. aureus specic genes, Ala-tRNA transferase, dna I, and glyS, as positive controls, and Arabidopsis thaliana specic genes, Lhb1B2, LHCP, and psbP, as negative controls. Adding the three negative control genes, we used a total of 224 genes.

2.5. Printing Oligonucleotide probes were synthesized with an attached 50 -terminal linker (DNattachTM, Nisshinbo, Japan). The synthesized probes were printed onto carbodiimide slides (CarboStation-U; Nisshinbo) at a concentration of 120 mM in 0.1% sodium dodecyl sulfate and ArrayIts micro-spotting solution (TeleChem International Inc., Sunnyvale, CA). Three replicates were printed for each oligonucleotide probe using a contact microspotting robotic system developed by RIKEN. Complete microarray sections were printed on each slide glass in duplicate. The spots had an average diameter of 120 mm.

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2.6. Slide processing The printed slides were crosslinked with a UV cross-linker (XL-1000 Spectrolinker/B, Spectronics, Westbury, NY) at an exposure of 600 mJ/cm2 and washed twice by shaking in distilled water for 5 min each. The microarrays were then spin-dried at 480 g for 1 min and stored in the dark at 4 1C. 2.7. Quality control of oligonucleotide deposition SYBR green II staining was used to visualize individual DNA spots on glass slides before hybridization. Staining was performed according to a short technical report [37]. After staining, the slide was washed with TBE and airdried. Fluorescence images were taken with a scanner (DNAscopeTM V, Biomedical Photonics Inc., Waterloo, Canada) with lter sets appropriate for Alexa Fluor 488 dye or uorescein (FITC). Fluorescence signals from each spot were measured and analyzed with Macro View software (Biomedical Photonics Inc.). 2.8. Hybridization conditions A hybridization mixture containing 10 ng/ml Cy3-labeled DNA, 1 mg/ml yeast tRNA, 3.4 SSC, 4 TE, and 2% blocking solution (Roche Diagnostics, Grenzacherstrasse, Switzerland) in 150 ml total volume was used. The mixture was heated for 10 min at 95 1C, cooled for 1 min on ice, and centrifuged for 1 min at room temperature. The slides were hybridized in a hybridization station (HybStation, Genomic Solutions, Ann Arbor, MI) at 60 1C for 16 h. After hybridization, each slide was washed in the hybridization station with 2 SSC, 0.1% SDS for ve cycles at 50 1C, 1 SSC for ve cycles at 42 1C, and 0.2 SSC for ve cycles at 42 1C. Each cycle consisted of owing wash buffer for 20 s and static holding for 40 s. The slides were nally rinsed briey at room temperature in 0.2 SSC. Washed microarrays were dried by centrifugation at 480 g. 2.9. Microarray scanning Fluorescence images of the microarrays were obtained with a scanner (DNAscopeTM V). Fluorescence signals from each spot were measured and compared with Macro View software. 2.10. PCR amplication Gene-specic primers were used for amplication of individual staphylococcal genes. PCR was performed as described by Chongtrakool et al. [31]. 2.11. Sequencing Sequencing was carried out for some enterotoxin genes and coagulase genes using the protocol described by Watanabe et al. [38].

2.12. Nucleotide sequence accession numbers The accession numbers of sequences deposited in GenBank are listed in Supplementary Data. 3. Results 3.1. Design of microarray and oligonucleotide probes We selected 221 genes to characterize MRSA strains in terms of virulence, drug resistance, and evolutionary classication. A set of oligonucleotide probes was collected on a focused microarray to identify genes for (i) S. aureus specic proteins, (ii) staphylococcal proteins mediating antibiotic resistance and factors involved in their expression, (iii) putative virulence proteins and factors controlling their expression, and (iv) proteins produced by genomic islands, such as SCCmec and pathogenicity islands. The microarray was also designed to carry out typing of SCCmec using allele-specic gene probes. In designing the genome microarray, the variation of genome sequences during their rapid generation cycles is a matter that should not be overlooked. Using sequences that have been determined for staphylococcal genes, we designed 390 oligonucleotide probes in gene-specic regions. Some of the oligonucleotide probes may have some mismatches with the sequences of certain tested strains. In what follows, we explain the procedure for probe design. Among 221 genes selected, 164 existed commonly in the seven strains of S. aureus genomes: Of the 164 genes, 49 were commonly shared by the seven strains [9,3941]. For the 49 genes, we extracted sequences of 45 mer bases commonly present in the seven strains for use as probe sequences. In the case that there was no such completely common region, we designed probe sequence so that the number of mismatched bases among the seven strains was 4 or less. For the other 115 genes, the same approach was taken for strains that harbored orthologous genes. Apart from such independent genes, the identication of orthologs by a 45-mer specic probe was a complicated task; namely, two or more oligonucleotide probes had to be prepared to identify allotypes and gene families. In the case of coagulase proteins, multiple sequence alignment of 10 coagulase allotypes was performed to extract allotypespecic sequences from closely related sequences. We designed probes in specic regions so that eight or more bases were different from the other types of coagulases in nucleotide sequence. For the coagulase family, we rst designed type-specic probes in the D1 region, which had the greatest sequence variety among coagulases [38]. However, the design of the type-specic probes in the D1 region was not sufcient to identify each coagulase type. To show an example, a probe designed for type I coagulase possessed a homologous region in the type X coagulase sequence with a difference of only one base. The design of type-specic probes in the D2 region was not sufcient, either. A probe designed for type IV coagulase possessed

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a homologous region in the type VIII coagulase sequence with a difference of only two bases. Thus, we adopted a multi-probe approach in which the coagulase type was determined only when the dual probes designed in D1 and D2 regions were positive. The same multi-probe approach was applied to the typing of other genes such as ccrA types, hsd types, integrase types, enterotoxin genes, and exfoliative toxin genes. 3.2. Typing strategy of SCCmec elements One purpose of the epidemiological microarray was to judge the types of SCCmec collectively. The principle of SCCmec typing is summarized in Fig. 1. SCCmec elements are composed of three important regions: mec gene complex, ccr gene complex, and J1 region. Conceptually, SCCmec types are assigned by determining mec class, ccr type, and J1 region type. The presence of the mecA gene was primarily monitored with two probes specic for that

gene. The presence of SCCmec elements was also checked with two other probes specic for IS431. Class A mec strains were identied by hybridization with the two probes specic for the gene, the two mecI probes for the membrane-spanning domain of the mecR1 gene, and the two probes for the penicillin-binding domain of the mecR1 gene. Similarly, class B mec strains were identied by the two probes specic for the membrane-spanning domain of the mecR1 gene and the three probes for the IS1272 gene. ccr gene complexes were typed by using sixteen probes specic for ccrA1, ccrA2; ccrA3, ccrA4, ccrB1, ccrB2, ccrB3, and ccrB4 and three probes specic for ccrC. The J1 region in the SCCmec element was further sub-typed by sixteen probes specic for eight specic sequences, including types I, IIa, IIb, III, IVa, IVb, IVc, and IVd. IS431 is an SCCmec-specic sequence. Although it is not directly correlated to the typing, we designed probes in this sequence as positive control for SCCmec elements. Probes

Fig. 1. Principle of SCCmec typing of S. aureus. (A) Schematic representation of the SCCmec element of S. aureus. Conceptually, SCCmec types were assigned by determining mec class, ccr type, and J1 region type. Gene-specic probes are shown as small rectangles. Six major SCCmec types are easily distinguishable: type I strains display type-1 ccr gene complex and class B mec gene complex; type II strains display type-2 ccr gene complex and class A mec gene complex; type III strains display type-3 ccr gene complex and class A mec gene complex; type IV strains display type-2 ccr gene complex and class B mec gene complex; type V strains display ccrC gene and mecA gene; and type VI strains display type-4 ccr gene complex and class B mec gene complex. Type II SCCmec subtypes, types IIa and IIb SCCmec, are distinguished by IIa and IIb specic sequences in the J1 region, respectively; and type IV SCCmec subtypes, types IVa, IVb, IVc, and IVd SCCmec, are distinguished by IVa, IVb, IVc, and IVd specic sequences in the J1 region, respectively. (B) Classication strategy of SCCmec elements. Determining whether mec is class A or B is the rst step of classication. The SCCmec class is determined by ccr type. Then, according to the J1 region sequence type, the subclass is determined. Types IIa, IIb, III, and IVc include Tn554 in their SCCmec sequences. MS: Membrane-spanning domain. PB: Penicillin-binding domain.

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related Tn554 genes such as tnpC were also added because it was interesting to check whether SCCmec always carried transposon Tn554. Using the 60 probes designed for typing, SCCmec became easily distinguishable according to the criteria presented in Fig. 1. 3.3. Conrmation of microarray functions with seven strains We validated the DNA microarrays using labeled genome targets obtained from the above seven strains. First, we set the hybridization temperatures in the range of 5565 1C. Discrimination was improved when the hybridization temperature was increased from 55 to 65 1C (data not shown). At 65 1C, however, some perfectly matched probes were identied as negative. In contrast, there were no false negatives at 60 1C. Thus, we concluded that 60 1C is the optimal temperature. In some instances, it was crucial to judge whether a strain carried a gene or not because spot signal intensities were not always separated well. Spots showing intermediate intensity signals should be assigned to either the positive or the negative group, although most spots were clearly classied into one of those two groups. Therefore, we uniquely determined a cut-off value as the threshold by drawing a histogram of appearance frequency of probes for their signal intensities as shown in Fig. 2. In Fig. 2(A), the histogram has two peaks in the lower and higher intensity sides. The midpoint of those peaks is identied as the threshold. When the signal intensity on a spot is larger than

the threshold, that spot is regarded as positive. On the other hand, when it becomes smaller than the threshold, that spot is regarded as negative. Fig. 2(B) presents examples of histograms, in which such peak separation is somewhat unclear. Even such cases, we applied the same criteria of the threshold which is set at the midpoints of the lower and higher peaks. Although other rejection methods were tested, such as negative control +2SD or positive control 2SD, they resulted in many false-positive results. Finally, we concluded that this simple method using the midpoint threshold was the most reliable and accurate according to a blind test with some of the fully sequenced strains (data not shown). At the beginning of this study, we used the one-probe one-gene array design. The accuracy of overall genotyping was 98.2% on average (data not shown). Most of the genotyping failures were due to false-positive results. We analyzed how many nucleotide differences the probes can detect. Probes containing 4 or less nucleotide mismatches were detected positively. Probes with eight or more nucleotide mismatches gave positive signals with a probability of less than 10%. The probability of showing positive signals with probes containing 58 nucleotide mismatches was in between. With such moderate stringency in preparation to the variance of genome sequence, occasional cross-reacting spots occurred, such as a sea spot that cross-reacted with other types of enterotoxin genes because of sequence similarity. Finally, we reduced the probability of a false-positive result by designing two or

120 100 80 60 40 20 0 1.5 2 2.5 3 3.5 4 4.5 5

70 60 50 40 30 20 10 0 1.5 2 2.5 3 3.5 4 4.5 5

Fig. 2. Typical histograms of microarray signals. The distribution of the appearance frequency of probes is presented as a function of the common logarithmic intensity of their signal intensities. Symbols , &, K and J present the results of strains, Mu50, NCTC8325,COL and N315, respectively. Arrows indicatJCSCe the threshold points for the positive or negative judgments of probes.

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more probes for each gene. By using multiple oligonucleotide probes to detect genes, the interference of the small number of cross-reacting spots were minimized in genotype identication. Finally, the microarray-based assay could accurately detect all 164 genes with 100% accuracy. The results of microarray analysis of all 221 genes for the seven strains are shown in Supplementary Data. The results demonstrate the high performance of the microarray in discriminating the genes. To evaluate the reproducibility and reliability of hybridization signals, three independent operators were labeled with genomic DNA of strain N315 and hybridized. Pearsons correlation coefcients of 0.930.97 indicate the reproducibility of hybridization between replicate microarrays. The genotyping results of the three independent operators were accurate and had 100% specicity. The correlation coefcients and the genotyping results demonstrated that the microarray is a sufciently accurate system. 3.4. Comparison of microarray results with PCR amplication results for 18 chosen genes To further evaluate the microarray, we studied genes that are absent from the seven genome-sequenced strains, but were detected in another 27 strains (Table 2). The genes were potentially transmissible drug resistance genes, toxin genes, or several genes of phylogenetic interest. We performed PCR to conrm the absence or presence of the genes in the 27 strains. Toxin genes eta, etb, etd, sec1, seb, sed, see, and sej were detected in strains JSCS6306, JCSC3057, TY114, Ku, JCSC1469, JCSC6490, JCSC6491, and JCSC6492, respectively. Drug resistance genes tetK, ermB, msrA, aphA3, and fosB were detected in strains JCSC1469, JCSC4519, JCSC4607, JCSC6084, and JCSC6489, respectively. Enterococcus-derived drug resistance genes vanA and vanB were found in E. faecium strains 1658 and 1732, respectively (Table 2). Capsule genes capH1, capI1, and capJ1 were carried by strain JSCS6488. We further chose another 14 genes, such as mecA, tnpA, tnpB, tnpC, capsulerelated genes, and enterotoxin genes, and a total of 32 genes were compared in terms of DNA microarray and PCR amplication results. Amplicons were designed so that they included regions of oligonucleotide probes in preparation for genome sequencing when necessary. The results obtained by microarray analysis agreed well with those by PCR analysis. We found a 99.4% concordance rate between them (859 of 864 possible genotypes). From the genotype data in Table 2, however, some discrepancies were observed; ve cases in total among tnpC, capK5, capK8, and etb genes. In strain Ku, tnpC showed positive hybridization signal but no PCR amplication signal. In strain JCSC6489, etb gave an arraypositive signal although the intensity was weak and the signal existed in the critical zone for positive judgment, whereas it was not amplied by PCR. Nonspecic hybridization may give a false-positive result because the strains have homologous sequences as probes. In addition

to such genes, some genes that gave false-positive resulted, which may be attributed to cross hybridization. In the case of capK5 in strain L20A and strain JCSC6084 and capK8 in strain JCSC4711, only weak hybridization signals were detected and no conclusive judgment could be made. On the other hand, it was difcult to judge PCR amplication signals of capsule genes capK5 and capK8, because they did not show strong amplication signals. The hybridization signals of these two genes, however, were detected accurately in the seven sequenced strains with sufcient signal intensities. 3.5. Comparison of coagulase typing by microarray with that by serological test In the case of coagulases, we compared microarray results with the results of serological test. As mentioned earlier, two probes were prepared for each coagulase. The results are presented in Table 3. Coagulase types determined with the microarray agreed with their phenotypes determined by the serological test. However, there were two strains whose coagulase types could not be judged by the microarray-based assay. Type V coagulase of strain (serotype) JCSC2167 gave negative signal for one of the type V probes. On the other hand, neither of the two probes was showed positive for type VI coagulase of strain (serotype) JCSC1469. To elucidate the reason for the discrepancy, the sequences of coagulase genes were analyzed for those two strains. It was found that the positive-signal probe for type V coagulase presented only one base mismatch against the sequence of JCSC2167 coagulase, while the negative-signal probe exhibited nine base mismatches against the corresponding sequence. Regarding type VI coagulase of JCSC1469, both probes exhibited 14 base mismatches against the coagulase sequence. It is reasonable that mismatches exceeding nine bases would not give positive hybridization signals. Therefore, sequence variations that could not be detected by the present serological test were detected by the microarray-based assay. Sequencing of the coagulase gene revealed that the two strains, JCSC2167 and JCSC1469, could not be classied into any known genotypes and may therefore represent novel genotypes. Notably, the sequence correspondence between the untypeable coagulase gene of JCSC2167 and type V coagulase gene was found to be approximately 88.3%. On the other hand, the coagulase gene sequence of JCSC1469 was more similar to type IV (79.8%) or IX (81.0%) than to type VI (74.8%) coagulase gene sequence. The probe set for microarray typing detected the sequence differences, although the phenotypes were the same. 3.6. Validation of microarray-based assignment of SCCmec elements The SCCmec hybridization proles of 34 strains that include strains used in the PCR verication are shown and

Table 2 Comparison of array hybridization with PCR amplication for 32 genes

Gene strain tnpA tnpB tnpC mecA *tetK *aphA3 *vanA *vanB *ermB *msrA *fosB capH5 capI5 capJ5 capK5 capH8 capK8 *capH1 *capI1 *capJ1 sea L20A 1658 1732 TY114 JCSC1469 JCSC2167 JCSC4469 JCSC4519 JCSC4607 JCSC6084 JCSC6306 JCSC6488 JCSC6489 JCSC6490 JCSC6491 JCSC6492 85/2082 JCSC6054 JCS-C3057 JCSC1978 MR108 Wis Ku JCSC4711 JCSC4712 NCTC10442 85/3907 **MW2 **N315 +/+ / / / / / +/+ / / +/+ / / / / / / +/+ / +/+ / +/+ / / / / / +/+ +/+ +/+ +/+ / / / / / +/+ / / +/+ / / / / / / +/+ / +/+ / +/+ / / / / / +/+ +/+ +/+ +/+ / / / / / +/+ / / +/+ / / / / / / +/+ / +/+ / +/+ / +/ / / / +/+ +/+ +/+ +/+ / / / +/+ +/+ +/+ / / +/+ / / / / / / +/+ +/+ +/+ +/+ +/+ +/+ / / / +/+ +/+ +/+ +/+ / / / / +/+ / / / / / / / / / / / +/+ / / / / / / / / +/+ +/+ / / +/+ / +/+ / / +/+ / / / +/+ / / / / / / / / / / / / / / / / +/+ / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ +/+ / / / / +/+ / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / +/+ / / +/+ / +/+ +/+ +/+ +/+ / / / +/+ +/+ / +/+ / +/+ / +/+ / / +/+ / / +/+ / / +/+ +/+ / / +/+ / +/+ +/+ +/+ +/+ / / / +/+ +/+ / +/+ / +/+ / +/+ / / +/+ / / +/+ / / +/+ +/+ / / +/+ / +/+ +/+ +/+ +/+ / / / +/+ +/+ / +/+ / +/+ / +/+ / / +/+ / / +/+ / +/+ +/+ +/+ / / +/+ / +/+ +/+ +/+ +/+ /+ / / +/+ +/+ / +/+ / +/+ / +/+ / / +/+ / / +/+ / +/+ +/+ / / / / / / / / / +/+ +/+ +/+ / / +/+ / +/+ / +/+ / +/+ +/+ / +/+ +/+ / +/+ / / / / / / / / / / / +/+ +/+ +/+ / / +/+ / +/+ / +/+ / +/+ +/+ / +/+ +/+ / +/+ / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / +/+ / / / / / / / / +/+ / / / +/+ / / +/+ / / / / / / / / / +/+ +/+ / *seb *sec1 *sed *see / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / +/+ / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / seg +/+ / / / +/+ +/+ +/+ / / / / / / / / / / +/+ / / +/+ +/+ / / / / / / +/+ seh / / / / / / / / / / / / / / / / / / / / / / +/+ / / / / / / sei +/+ / / / +/+ +/+ +/+ / / +/+ / / / / / / / +/+ / / +/+ +/+ +/+ / / / / +/+ +/+ *sej / / / / / / / / / / / / / +/+ / +/+ / / / +/+ / / / / / / / / / *eta / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / *etb *etd / / / / / / / / / / / / +/ / / / / / +/+ / / / / / / / / / / / / / +/+ / / / / / / / / / / / / / / / / / / / / / / / / /

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+/+: both of array hybridization signal and PCR bands were positive. /+: array signals were negative and PCR bands positive. +/: vice versa. /: both of hybridization and PCR signals were negative. Bold: the bold emphasizes the inconsistencies between the array and PCR results. *: genes absent in the seven genome sequenced strains. **: genome sequenced strains as references.

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J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 113 9

compared in Table 4. Four strains previously determined as MSSA were conrmed to have neither mecA nor IS431. All the other strains were positive for mecA and IS431, both of which were the positive controls of SCCmec elements. Most of MRSA typing results agreed well with conventional typing results. Three additional ndings were obtained from the microarray analysis. Type II SCCmec strain MRSA252 was positive for both types IIa and IVc specic regions. To explain this result, we rechecked the fully determined E-MRSA252 genome sequence and found that it possessed the same sequence as type IVc specic gene (2121 bp) and percentage matching of the overlap region was equal to 100%. Thus, the microarray correctly showed that the strain possesses types IIa and IVc specic regions. Tn554 is known to be inserted only in types IIa, IIb, III, IVc, and IVd SCCmec [4]. In the microarray analysis, the ve genes related to Tn554 showed positive signals for the above ve types of strains. It was found that Tn554 genes gave also positive signals even in strains L20A and JCSC6084 that have type I SCCmec. We found concordance of DNA microarray data with PCR amplication data of the ve genes of those two strains. In strain Ku, the tnpC gene was detected, although Ku is an MSSA. As pointed out in Section 3.4, PCR gave a negative result. 4. Discussion We have developed a microarray-based assay for the detection and identication of staphylococcal genes. The assay was based on random-primer labeling of the whole genome region. We validated the assay with three different approaches. First, using labeled genome targets of strain N315, the microarray results were proven to be highly reproducible when tested with three different operators at the optimal temperature for hybridization. Second, we validated the assay using DNAs from seven strains whose entire nucleotide sequences have been determined. The microarray showed 100% genotyping accuracy for the strains and detected all the 164 genes present in the strains. Thus, it was conrmed that the use of multiple oligonucleotide probes for the detection of a staphylococcal gene had the advantages of increased specicity and sensitivity. Third, 18 genes that were absent from the seven strains but were present in some of the tested strains were used to validate the assay system by comparison with PCR amplication results. For the 18 genes, PCR amplication data were highly consistent with microarray hybridization data, and there was only one case of discrepancy. We obtained 99.5% concordance rate between the two methods. However, for the other 14 genes tested at the same time, which were present in the seven strains, ve cases of discrepancy were found. Using 34 strains in total, we validated 182 genes, and the total genotyping accuracy of the 182 genes was 97.8% (178/182 genes). For the gene judgments in total with 164 gene probes of the seven strains

and 32 gene probes of the 27 strains, the concordance rate was as high as 99.8% (2007/2012 judgments). We adopted 45 mer oligonucleotide probes in the microarray because they present two advantages. One is sequence sensitivity, as the 45 mer probes can detect small sequence differences among homologous types and gene families. For example, the probes for coagulase typing detected such differences. Coagulase types determined with the microarray coincided with their phenotypes determined by the serological test except for two strains, JCSC2167 and JCSC1469. For the sequence of JCSC2167 coagulase, the probe that gave no hybridization signals exhibited nine base mismatches against the corresponding sequence. Regarding type VI coagulase of JCSC1469, the two probes that gave no hybridization signals possessed 14 base mismatches against the corresponding coagulase sequence. Overall, the microarray detected small sequence differences and unexpectedly frequent variations of genome sequences even within a typed family of coagulase. Serological typing seems to be insensitive to small variations detectable by DNA-based methods. The other advantage of the microarray method is that it enables us to nd consensus sequences within the same gene. Although such consensus sequences do not enable probes to identify 4 or less base mismatches among the same gene of different strains, they can indicate which family the strains belong to. Using such a consensus sequence probe, we can avoid the situation observed in JCSC1469 where all the coagulase probes gave negative results and the existence of coagulase gene itself could not be determined. Five cases of discrepancy were observed between microarray and PCR amplication results. In the case of tnpC in strain Ku, the hybridization signal was signicantly strong so that the possibility of misrecognition or misjudgment was low. Mutations in the primer regions may cause failure of amplication. The hybridization results of tnpC in the seven genome-sequenced strains were consistent. Ku is an MSSA but recently, it has been pointed out that some tnpC genes exist independently of SCCmec [4]. There also exists a gene that may be responsible for a false-positive result. For the etb gene, nonspecic hybridization may produce a false-positive result because the strains have a homologous sequence to the oligonucleotide probes. In such a case, only weak hybridization signals are produced, which are found in the valley between positive and negative hybridization signals in a histogram. In the cases of capsule genes capK5 and capK8, a possibility of crosstalk among PCR primers and capsule gene family can be considered, in addition to cross hybridization in the microarray. Collective investigation with microarray probes was also found to be effective in the analysis of the structure of SCCmec. Here, we followed the present criteria used for classication: namely, the structure of the mecA complex (mecA gene with upstream regulatory region), the nature of ccr gene complexes, and the function associated with the insertion and excision of the mec element at the specic

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10 J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 113 Table 3 Results of array-based assignment of coagulases and their corresponding phenotypes Phenotype starin I JCSC3057 II III IV N315 NTCT8325 MRSA252 V JCSC6306 V JCSC2167 VI JCSC1469 VII VIII MW2 Ku IX JCSC4711 X JCSC4712

Coagulase type (region) I(D1) + I(D2) + II(D1) II(D2) III(D1) III(D2) IV(D1) IV(D2) V(D1) V(D2) VI(D1) VI(D2) VII(D1) VII(D2) VIII(D1) VIII(D2) IX(D1) IX(D2) X(D1) + X(D2) Coagulase type I determined by the array

+ + + II

+ + III

+ + + + IV

+ + V

+ +

+ + VII

+ + VIII

+ + + IX

+ + + X

+: the array hybridization signals were considered positive signals. Coagulase types were uniquely determined by judgment whether their two type-specic probes were positive or not.

chromosomal site of orfX [36]. The microarray-based method provided maximum resolution for various structural variants of the SCCmec element that we have identied in association with various MRSA epidemiological types. This method enabled identication of not only the mecA complex but also the ccr gene complex: it was capable of discriminating six major SCCmec types and subtypes (Table 4). Several PCR-based methods are available for S. aureus typing [42]. Recently, an S. aureus typing method that utilizes multiplex PCR was reported [43]. These PCR methods are based on combinations of gene-specic primers or a combination of universal forward primers and specic reverse primers. One drawback of all the present PCR-based methods is that they do not exhibit high-throughput performance. As several separate reactions are required in order to distinguish several genes, the diagnostic process is time consuming and laborious. One problem in analyzing the relationship between the presence of a specic gene (or a combination of genes) and staphylococcal infection is that it is necessary to characterize gene combinations of infectious strains on a large scale. DNA microarray provides a straightforward solution to this problem. In one leading study, PCR products were used as probes [44,45]. In microarrays that use PCR products as probes, specic gene regions for the identication of homologous gene families and types cannot be selected as freely as when oligonucleotide probes are used because the lengths of PCR products used as

probes usually exceed 100 bases. In addition, use of PCR products as probes would result in low hybridization specicity compared with use of oligonucleotide probes because their lengths would easily cause cross hybridization. As has been discussed, the use of oligonucleotide probes instead of PCR products as probes enables us to select specic probe regions for the identication of homologous gene families and types, facilitating and improving the microarray performance, since no PCR and no template DNA of reference strains are necessary. Two kinds of oligonucleotide probe microarrays were mentioned in the Introduction [29,30]. One was a whole genome microarray covering approximately 90% of three S. aureus strain genomes [29]. The other one focused on 383 selected genes [30]. To design those probes, basic sequences were adopted from Mu50, N315, MW2 or COL. In contrast to single-color hybridization presented in this paper, two-color comparative hybridization was applied with a sample mixture of the above strains as reference. From a practical standpoint, microarrays for wide-scale epidemiologic use should be cost-effective. In this regard, the DNA microarray described herein, which uses a small number of genes with single-color labeling, is preferable. Due to space limitations, we briey discussed the genes involved in epidemiological classication, such as coagulase and SCCmec genes. To determine the clinical signicance of genes encoding virulent proteins, we have started a separate study of clinical MRSA strains to

Table 4 Results of array-based assignment of SCCmec elements Strain COL L20 N315 MRSA252 JCSC3057 85/ 2082 85/ 3907 MW2 JCSC1978 MR108 JCSC4469 WIS JCSC6054 MSSA476 Ku Strain1658 Strain1732 JCSC4519

NCTC10442 JCSC6084 Mu50

JCSC2167 JCSC1469

NCTC8325 JCSC4711 JCSC4712

JCSC4607 JCSC6306 JCSC6488 JCSC6489 JCSC6490 JCSC6491 JCSC6492 TY114 MSSA E. faecalis E. faecalis Not tested

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SCCmec allotype determined by other studies Array judgment mecA gene complex ccr complex J1 region Genes in Tn554 TnpA in Tn554 TnpB in Tn554 TnpC in Tn554 ermA ant(9)(spectinomycin) IS431*

Not tested II a

II a

II b

III

IV a

IV b

IV c

IV d

VI

MSSA

I B Type 1 I

I0 B Type 1 I

II a A

II a0 A

II b A Type 2 II b

III A

IV a B

IV b B Type 2 IV b

IV c B

IV d B

VI

No SCC None None None

Type Type 2 2 II a II a and IV c + + + + + + + + + + + +

Type3 Type 2 III IV a

Type 2 Type 2 IV c IV d

mecA B only ccrC Type 4 V VI

No No SCC SCC None None None None None None

No SCC None None None

No SCC None None None

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+ + + + + +

+: array hybridization signals were positive. Bold: the bold represents the genes amplied using PCR. *IS431 is the SCCmec specic sequence. We designed probes in this sequence as the positive control for SCCmec elements.

11

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12 J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 113 vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Ch 2006;50:42838. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Oguchi A, et al. Genome and virulence determinants of high virulence communityacquired MRSA. Lancet 2002;359:181927. Dryla A, Prustomersky S, Gelbmann D, Hanner M, Bettinger E, Kocsis B, et al. Comparison of antibody repertoires against Staphylococcus aureus in healthy individuals and in acutely infected patients. Clin Diagn Lab Immun 2005;12:38798. Akiyama H, Hamada T, Huh WK, Yamasaki O, Oono T, Fujimoto W, et al. Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in skin lesions of bullous impetigo, atopic dermatitis and pemphigus foliaceus. Br J Dermatol 2003;148:52632. Zschock M, Nesseler A, Sudarwanto I. Evaluation of six commercial identication kits for the identication of Staphylococcus aureus isolated from bovine mastitis. J Appl Microbiol 2005;98:4505. Jorgensen JH, Crawford SA. Assessment of two commercial susceptibility test methods for determination of daptomycin MICs. J Clin Microbiol 2006;44:21269. Safdar N, Narans L, Gordon B, Maki DG. Comparison of culture screening methods for detection of nasal carriage of methicillinresistant Staphylococcus aureus: a prospective study comparing 32 methods. J Clin Microbiol 2003;41:31636. Bergdoll MS. Importance of staphylococci that produce nanogram quantities of enterotoxin. Zentralbl Bakteriol 1995;282:16. Casey AL, Worthington T, Caddick JM, Hilton AC, Lambert PA, Elliott TS. RAPID for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection. J Infection 2006;52:2829. Boerema JA, Clemens R, Brightwell G. Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus. Int J Food Microbiol 2006;107:192201. Vivoni AM, Moreira BM. Application of molecular techniques in the study of Staphylococcus aureus clonal evolutiona review. Mem I Oswaldo Cruz 2005;100:6938. Velasco D, del Mar Tomas M, Cartelle M, Beceiro A, Perez A, Molina F, et al. Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. Antimicrob Agents Ch 2005;55:37982. Thorell EA, Selvarangan R. Methicillin-resistant Staphylococcus aureus (MRSA) in healthy children: risk factor (RF) analysis and pulsed eld gel electrophoresis (PFGE) of colonizing and invasive strains. Pediatr Res 2006;60:498. Stephens AJ, Huygens F, Inman-Bamber J, Price EP, Nimmo GR, Schooneveldt J, et al. Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J Med Microbiol 2006;55:4351. Schena M, Davis RW. DNA microarrays: a practical approach (practical approach series). In: Schena M, editor. Genes, genomes and chips. Oxford: Oxford University Press; 1999. p. 115. Takahashi M, Kondoh Y, Tashiro H, Koibuchi N, Kuroda Y, Tashiro T. Monitoring synaptogenesis in the developing mouse cerebellum with an original oligonucleotide microarray. J Neurosci Res 2005;80:77788. Cui L, Lian JQ, Neoh HM, Reyes E, Hiramatsu K. DNA microarray-based identication of genes associated with glycopeptide resistance in Staphylococcus aureus. Antimicrob Agents Ch 2005;49:340413. Nubel U, Schmidt PM, Reiss E, Bier F, Beyer W, Naumann D. Oligonucleotide microarray for identication of Bacillus anthracis based on intergenic transcribed spacers in ribosomal DNA. FEMS Microbiol Lett 2004;240:21523. Booth SA, Drebot MA, Martin IE, Ng LK. Design of oligonucleotide arrays to detect point mutations: molecular typing of antibiotic resistant strains of Neisseria gonorrhoeae and hantavirus infected deer mice. Mol Cell Probe 2003;17:7784.

correlate the repertoire of virulence genes carried by the strains with the clinical picture of the infection caused by them. Collective gene assay with reliable and cost-effective microarrays will enable us to obtain information on the real clinical impact of staphylococcal virulence genes. 5. Conclusion In conclusion, the microarray-based assay described here is a powerful tool for the analysis of S. aureus strains. We applied this assay to 34 standard strains and validated the results by means of nucleotide sequencing and PCR tests. The overall concordance rate for 182 genes is 99.8%. The assay shows great potential for use in the highthroughput screening and accurate genotyping of staphylococcal genes, which are required for the epidemiological study of S. aureus clinical strains. Acknowledgments This work was supported by a grant from the Creation and Support Program for Start-ups from Universities of the Japan Science and Technology Agency. Appendix A. Supplementary data

[9]

[10]

[11]

[12]

[13]

[14]

[15] [16]

[17]

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.mcp. 2007.05.007.

[18]

[19]

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