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Molecular and Cellular Probes 22 (2008) 1–13

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Development and validation of microarray-based assay forepidemiological study of MRSA

Junnosuke Otsukaa,b, Yasumitsu Kondoha, Tomoyuki Amemiyaa, Akio Kitamurac,Teruyo Itoc, Satoshi Babac, Longzhu Cuic, Keiichi Hiramatsuc,

Tomoko Tashirob, Hideo Tashiroa,�

aProbing Technology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, JapanbDepartment of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Sagamihara, Kanagawa 229-8558, Japan

cDepartment 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 versatilepathogenicity in humans and animals. Enterotoxins,exfoliative toxins, and toxic shock syndrome toxins areknown to be responsible for various pathologies [1]. Thecoordinated action of several genes that are expressedunder the control of several regulatory systems also causesclinical symptoms [2,3]. S. aureus is one of the mostcommon causes of hospital infection. This has become agreat matter of concern because more than half of S. aureus

strains in hospitals are methicillin-resistant, or MRSAs,

e front matter r 2007 Elsevier Ltd. All rights reserved.

cp.2007.05.007

ing author. Tel.: +8148 467 9303; fax: +81 48 467 9300.

ess: htashiro@riken.jp (H. Tashiro).

which are resistant to most of the antibiotics used inhospitals [4,5]. In particular, MRSA strains that exhibitdecreased susceptibility to glycopeptides [6–8] have ex-tremely limited our choice of antibiotics for the treatmentof hospital-acquired S. aureus infections. Although themolecular events responsible for human pathogenesis arenot understood completely, many genes are presumed to beinvolved, and their allelic variations among strains areconsidered to influence the pathogenic potential and thedegree of drug resistance of each MRSA strain [9]. Analysisof variance of the gene repertoire among strains, therefore,would greatly enrich our knowledge of the mechanism ofS. 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.

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Even S. aureus strains that are deemed identical by suchphenotypic methods may actually be different when grownon various natural substrates and laboratory media [15]. Inparticular, serological methods cannot identify a largegroup of related proteins having significant antigenicsimilarities, such as enterotoxins.

In recent years, a variety of molecular genotypingtechniques have emerged to analyze S. aureus genediversity in a rapid and precise manner [16–19]. Pulse fieldgel electrophoresis (PFGE) is popularly used as a tool formolecular epidemiology [20]. However, it presents only therelatedness of strains and does not convey any informationon the content of the genome or the phenotypic potentialof strains. In addition, its procedure is complex and timeconsuming. Multilocus sequence typing (MLST) wasintroduced as a staphylococcal typing method [21].Sequences of the internal fragments of seven housekeepinggenes are determined for each isolate, thereby defining thespecific alleles for each locus. However, although MLSTcan classify strains into evolutionarily relevant groups, itdoes not provide any biologically or medically pertinentinformation.

In view of the above situation, the microarray system isexpected to be a powerful epidemiological tool because itcan collectively detect thousands of genes or target DNAsequences on a single glass slide [22]. Although themicroarray methods used in most studies have focused onthe study of gene expression [23,24], they can be adoptedfor the DNA-based typing of specific pathogenic bacterialstrains [25–28]. Two oligonucleotide microarrays forS. aureus have been constructed [29,30]. StaphChipcomprises 5427 probes covering approximately 90% ofthree S. aureus strain genomes [29], and is effective forwhole-genome analysis. However, from a practical stand-point, probes should be made for selected genes becausethe synthesis of many probes is generally a very expensivetask. The other microarray consists of 383 probes coveringvirulence genes [30]. However, the sensitivity and reliabilityof the oligonucleotide microarray have not been proven sofar. More trials are needed to design a medically usefulDNA microarray having sufficiently high precision andreliability.

Here, we have designed and tested a DNA microarraywith gene-specific oligonucleotide probes to detect not onlyimportant genes but also the types of genomic islandscarried by each MRSA strain. A set of probes wereprimarily designed to identify virulence genes. The micro-array also contained probes designed for the detection ofgenomic islands. The most typical and important genomicisland is SCCmec element, which carries drug resistancegenes such as the methicillin resistance gene, mecA [31–33].The acquisition of methicillin resistance occurs viahorizontal transfer of SCCmec from other staphylococcalstrains. SCCmec has various types and subtypes and thedetection of SCCmec type is expected to provide informa-tion relating to the origin of the MRSA strain. Forexample, SCCmec carried by health-care-associated MRSA

(H-MRSA) differs from that carried by community-acquired MRSA (C-MRSA) [34]. Therefore, SCCmec

typing is essential for the characterization of clinicalMRSA strains.Another important genomic island is pathogenicity

island. High throughput gene sequencing of severalS. aureus genomes has revealed the presence of largechromosomal regions carrying many virulence genes [35].Representative pathogenic islands, nuSaa and nuSab, arefound in practically all clinical strains, but in distinct allelicforms. The detection of the allelic types of the islands,therefore, would give us information on the evolutionaryrelationship of MRSA strains [36].We report here the development of a new DNA

microarray system that conveys both virulence andevolutionary information of MRSA strains.

2. Materials and methods

2.1. Bacterial strains

Bacterial strains used in this experiment are listedin Table 1. We selected 32 S. aureus strains and twoE. faecalis strains.

2.2. Total DNA preparation

Total cellular DNA was extracted and purified witha MagPreps bacterial genomic DNA kit (Novagen,San Diego, CA), according to the manufacturer’sinstructions. The extracted DNA was fragmented with asonicator (VP-5S homogenizer, TAITEC, Japan) to anaverage size of 1000 bp. Concentration and purity ofgenomic DNA in the prepared samples were determinedby measuring the absorbance at 260 and 280 nm with aspectrophotometer (Gene Spec V, Naka Instruments,Japan).

2.3. DNA labeling

Fragmented DNA (1 mg) was labeled with chemicallyreactive nucleotide analog (aminoallyl-dUTP) by random-primer labeling (BioPrimes plus array CGH genomiclabeling system, Invitrogen, Carlsbad, CA) to generateaminoallyl-labeled DNA. Then, the aminoallyl-labeledDNA was coupled with Cy3-N-hydroxysuccinimide esters(GE Healthcare Bio-Sciences Corp., Piscataway, NJ).Cy3-labeled DNA was purified with Ultra PCR clean-upkit (ABgene, Rochester, NY) following the manufacturer’sinstructions. The purified DNA was stored at �20 1C untiluse in hybridization.

2.4. Sequence design of specific oligonucleotide probes

A total of 390 oligonucleotide probes were designed for221 selected genes and DNA regions. The first considera-tion was to choose regions specific to each gene and the

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Table 1

Strains used in this study

Strain ORFs used for veryfying microarray Coagulase

serotype

SCCmec

type

Source or references

Genome sequenced S. aureus strains

N315 102 ORFs II IIa Kuroda et al., 2001 [39]

Mu50 108 ORFs II IIa Kuroda et al., 2001 [39]

MW2 96 ORFs VII IVa Baba et al., 2002 [9]

NCTC8325 73 ORFs III MSSA http://www.genome.ou.edu/staph.html

MRSA252 97 ORFs IV IIa Holden et al., 2004 [40]

MSSA476 82 ORFs VII MSSA Holden et al., 2004 [40]

COL 81 ORFs III I Gill et al., 2005 [41]

S. aureus strains used for validation of specific genes or gene alleles

TY114 Exfoliative toxin D (ETD) Not tested Not tested Provided by M. Sugai, Hiroshima Univ., Japan

ZM Exfoliative toxin A (ETA) Not tested Not tested Provided by Y. Yoshizawa, Jikei Medical Univ.,

Japan

030-1 Exfoliative toxin B (ETB) I Iib Hisata et al., 2005 [6]

196E Staphylococcal enterotoxin (sea, sed,

selj, selr)

Not tested Not tested Provided by K. Omoe, Iwate Univ., Japan

FRI326 Staphylococcal enterotoxin (see, selq) Not tested Not tested Provided by K. Omoe, Iwate Univ., Japan

Fukuoka5 Staphylococcal enterotoxin (selj, selr) Not tested Not tested Provided by K. Omoe, Iwate Univ., Japan

M Type-I capsule (capH1, capI1, capJ1) Not tested Not tested Provided by J. Lee, Hervard Univ. USA

NCTC10442 Type-I SCCmec, tetK IV I Ito et al., 2001 [46]

85/3907 Type-III SCCmec, tetK, ermB IV III Ito et al., 2003 [32]

JCSC1469 Type-IVa SCCmec, type VI coagulase VI IVa Okuma et al., 2002 [47]

JCSC2167 Type-IVa SCCmec, type V coagulase V IVa Okuma et al., 2002 [47]

8/6-3P Type-IVb SCCmec III IVb Ma et al., 2002 [34]

81/108 Type-IVc SCCmec IV IVc Ma et al., 2006 [48]

JCSC4469 Type-IVd SCCmec Not tested IVd Ma et al., 2006 [48]

85/2082 Type-III SCCmec, tetK Not tested III Ito et al., 2001 [46]

WIS Type-V SCCmec I V Ito et al., 2003 [32]

HDE288 Type-VI SCCmec Not tested VI Provided by H. de Lencastre, Rockfeller, USA.

Ku Type VIII coagulase VIII MSSA Watanabe et al., 2005 [38]

JCSC4711 Type IX coagulase IX MSSA Watanabe et al., 2005 [38]

JCSC4712 Type X coagulase X MSSA Watanabe et al., 2005 [38]

NCTC8325(pI258) ermB Not tested Not tested Provided by Y. Nakajima, Japan

NCTC8325(pEP2104) msrA Not tested Not tested Provided by M. Matsuoka, Japan

MSC-3228 aac(60)-aph(200), aphA-3 Not tested Not tested Provided by T. Ida, Meiji Seika Kaisha, Japan

RN4220/pMF151 fosB Not tested Not tested Provided by T. Ida, Meiji Seika Kaisha, Japan

L20A Conjugative plasmid NT Not tested Provided by N. Noguchi, Tokyo phamacuetical

Univ., Japan

E. faecalis strains used for validation

1658 VanA This study

1732 VanB This study

J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 1–13 3

second was to avoid secondary structures with the useof Oligo 6.0 software (Molecular Biology Insights,Cascade, CO). The leading guidance was (i) 45 bp probein length, (ii) 8278 1C in melting temperature (Tm), and(iii) o�2.1 kcal/mol for hairpins. The specificity of thedesigned oligonucleotide probes was assessed with GENE-TYX-MAC software (Molecular Biology Insights, Cas-cade, CO) using the whole genome sequences of sevenstaphylococcal strains available in GenBank.

We selected S. aureus specific genes, Ala-tRNA transfer-ase, dna I, and glyS, as positive controls, and Arabidopsis

thaliana specific genes, Lhb1B2, LHCP, and psbP, asnegative controls. Adding the three negative control genes,we used a total of 224 genes.

2.5. Printing

Oligonucleotide probes were synthesized with anattached 50-terminal linker (DNattachTM, Nisshinbo,Japan). The synthesized probes were printed ontocarbodiimide slides (CarboStation-U; Nisshinbo) at aconcentration of 120 mM in 0.1% sodium dodecylsulfate and ArrayIts micro-spotting solution (TeleChemInternational Inc., Sunnyvale, CA). Three replicates wereprinted for each oligonucleotide probe using a contactmicrospotting robotic system developed by RIKEN.Complete microarray sections were printed on each slideglass in duplicate. The spots had an average diameterof 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 anexposure of 600mJ/cm2 and washed twice by shaking indistilled water for 5min each. The microarrays were thenspin-dried at 480� g for 1min and stored in the dark at 4 1C.

2.7. Quality control of oligonucleotide deposition

SYBR green II staining was used to visualize individualDNA spots on glass slides before hybridization. Stainingwas performed according to a short technical report [37].After staining, the slide was washed with TBE and air-dried. Fluorescence images were taken with a scanner(DNAscopeTM V, Biomedical Photonics Inc., Waterloo,Canada) with filter sets appropriate for Alexa Fluor 488dye or fluorescein (FITC). Fluorescence signals from eachspot were measured and analyzed with Macro Viewsoftware (Biomedical Photonics Inc.).

2.8. Hybridization conditions

A hybridization mixture containing 10 ng/ml Cy3-labeledDNA, 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 mixturewas heated for 10min at 95 1C, cooled for 1min on ice, andcentrifuged for 1min at room temperature. The slides werehybridized in a hybridization station (HybStation, Geno-mic Solutions, Ann Arbor, MI) at 60 1C for 16 h. Afterhybridization, each slide was washed in the hybridizationstation with 2� SSC, 0.1% SDS for five cycles at 50 1C,1� SSC for five cycles at 42 1C, and 0.2� SSC for fivecycles at 42 1C. Each cycle consisted of flowing wash bufferfor 20 s and static holding for 40 s. The slides were finallyrinsed briefly at room temperature in 0.2� SSC. Washedmicroarrays were dried by centrifugation at 480� g.

2.9. Microarray scanning

Fluorescence images of the microarrays were obtainedwith a scanner (DNAscopeTM V). Fluorescence signalsfrom each spot were measured and compared with MacroView software.

2.10. PCR amplification

Gene-specific primers were used for amplification ofindividual staphylococcal genes. PCR was performed asdescribed by Chongtrakool et al. [31].

2.11. Sequencing

Sequencing was carried out for some enterotoxin genesand coagulase genes using the protocol described byWatanabe et al. [38].

2.12. Nucleotide sequence accession numbers

The accession numbers of sequences deposited inGenBank are listed in Supplementary Data.

3. Results

3.1. Design of microarray and oligonucleotide probes

We selected 221 genes to characterize MRSA strains interms of virulence, drug resistance, and evolutionaryclassification. A set of oligonucleotide probes was collectedon a focused microarray to identify genes for (i) S. aureus

specific proteins, (ii) staphylococcal proteins mediatingantibiotic resistance and factors involved in their expres-sion, (iii) putative virulence proteins and factors controllingtheir expression, and (iv) proteins produced by genomicislands, such as SCCmec and pathogenicity islands. Themicroarray was also designed to carry out typing ofSCCmec using allele-specific gene probes. In designing thegenome microarray, the variation of genome sequencesduring their rapid generation cycles is a matter that shouldnot be overlooked. Using sequences that have beendetermined for staphylococcal genes, we designed 390oligonucleotide probes in gene-specific regions. Some of theoligonucleotide probes may have some mismatches withthe sequences of certain tested strains. In what follows, weexplain the procedure for probe design.Among 221 genes selected, 164 existed commonly in the

seven strains of S. aureus genomes: Of the 164 genes, 49were commonly shared by the seven strains [9,39–41]. Forthe 49 genes, we extracted sequences of 45mer basescommonly present in the seven strains for use as probesequences. In the case that there was no such completelycommon region, we designed probe sequence so that thenumber of mismatched bases among the seven strains was 4or less. For the other 115 genes, the same approach wastaken for strains that harbored orthologous genes.Apart from such independent genes, the identification of

orthologs by a 45-mer specific probe was a complicatedtask; namely, two or more oligonucleotide probes had to beprepared to identify allotypes and gene families. In the caseof coagulase proteins, multiple sequence alignment of 10coagulase allotypes was performed to extract allotype-specific sequences from closely related sequences. Wedesigned probes in specific regions so that eight or morebases were different from the other types of coagulases innucleotide sequence. For the coagulase family, we firstdesigned type-specific probes in the D1 region, which hadthe greatest sequence variety among coagulases [38].However, the design of the type-specific probes in the D1region was not sufficient to identify each coagulase type.To show an example, a probe designed for type I coagulasepossessed a homologous region in the type X coagulasesequence with a difference of only one base. The design oftype-specific probes in the D2 region was not sufficient,either. A probe designed for type IV coagulase possessed

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a homologous region in the type VIII coagulase sequencewith a difference of only two bases. Thus, we adopted amulti-probe approach in which the coagulase type wasdetermined only when the dual probes designed in D1 andD2 regions were positive. The same multi-probe approachwas applied to the typing of other genes such as ccrA types,hsd types, integrase types, enterotoxin genes, and exfolia-tive toxin genes.

3.2. Typing strategy of SCCmec elements

One purpose of the epidemiological microarray was tojudge the types of SCCmec collectively. The principle ofSCCmec typing is summarized in Fig. 1. SCCmec elementsare composed of three important regions: mec genecomplex, 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 genewas primarily monitored with two probes specific for that

Fig. 1. Principle of SCCmec typing of S. aureus. (A) Schematic representation

assigned by determining mec class, ccr type, and J1 region type. Gene-specific

distinguishable: type I strains display type-1 ccr gene complex and class B mec

mec gene complex; type III strains display type-3 ccr gene complex and class A m

B mec gene complex; type V strains display ccrC gene and mecA gene; and type

Type II SCCmec subtypes, types IIa and IIb SCCmec, are distinguished by I

SCCmec subtypes, types IVa, IVb, IVc, and IVd SCCmec, are distinguished b

(B) Classification strategy of SCCmec elements. Determining whether mec is cla

by ccr type. Then, according to the J1 region sequence type, the subclass is

sequences. MS: Membrane-spanning domain. PB: Penicillin-binding domain.

gene. The presence of SCCmec elements was also checkedwith two other probes specific for IS431.Class A mec strains were identified by hybridization with

the two probes specific for the gene, the two mecI probesfor the membrane-spanning domain of the mecR1 gene,and the two probes for the penicillin-binding domain of themecR1 gene. Similarly, class B mec strains were identifiedby the two probes specific for the membrane-spanningdomain of the mecR1 gene and the three probes for theIS1272 gene. ccr gene complexes were typed by usingsixteen probes specific for ccrA1, ccrA2; ccrA3, ccrA4,ccrB1, ccrB2, ccrB3, and ccrB4 and three probes specificfor ccrC. The J1 region in the SCCmec element was furthersub-typed by sixteen probes specific for eight specificsequences, including types I, IIa, IIb, III, IVa, IVb, IVc,and IVd.

IS431 is an SCCmec-specific sequence. Although it is notdirectly correlated to the typing, we designed probes in thissequence as positive control for SCCmec elements. Probes

of the SCCmec element of S. aureus. Conceptually, SCCmec types were

probes are shown as small rectangles. Six major SCCmec types are easily

gene complex; type II strains display type-2 ccr gene complex and class A

ec gene complex; type IV strains display type-2 ccr gene complex and class

VI strains display type-4 ccr gene complex and class B mec gene complex.

Ia and IIb specific sequences in the J1 region, respectively; and type IV

y IVa, IVb, IVc, and IVd specific sequences in the J1 region, respectively.

ss A or B is the first step of classification. The SCCmec class is determined

determined. Types IIa, IIb, III, and IVc include Tn554 in their SCCmec

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related Tn554 genes such as tnpC were also added becauseit was interesting to check whether SCCmec always carriedtransposon Tn554. Using the 60 probes designed fortyping, SCCmec became easily distinguishable accordingto the criteria presented in Fig. 1.

3.3. Confirmation of microarray functions with seven strains

We validated the DNA microarrays using labeledgenome targets obtained from the above seven strains.First, we set the hybridization temperatures in the range of55–65 1C. Discrimination was improved when the hybridi-zation temperature was increased from 55 to 65 1C (datanot shown). At 65 1C, however, some perfectly matchedprobes were identified as negative. In contrast, there wereno false negatives at 60 1C. Thus, we concluded that 60 1Cis the optimal temperature.

In some instances, it was crucial to judge whether astrain carried a gene or not because spot signal intensitieswere not always separated well. Spots showing intermedi-ate intensity signals should be assigned to either thepositive or the negative group, although most spots wereclearly classified into one of those two groups. Therefore,we uniquely determined a cut-off value as the threshold bydrawing a histogram of appearance frequency of probes fortheir signal intensities as shown in Fig. 2. In Fig. 2(A), thehistogram has two peaks in the lower and higher intensitysides. The midpoint of those peaks is identified as thethreshold. When the signal intensity on a spot is larger than

0

20

40

60

80

100

120

1.5 2 2.5 3

1.5 2 2.5 3

0

10

20

30

40

50

60

70

Fig. 2. Typical histograms of microarray signals. The distribution of the ap

logarithmic intensity of their signal intensities. Symbols ’, &, K and J pres

Arrows indicatJCSCe the threshold points for the positive or negative judgme

the threshold, that spot is regarded as positive. On theother hand, when it becomes smaller than the threshold,that spot is regarded as negative. Fig. 2(B) presentsexamples of histograms, in which such peak separation issomewhat unclear. Even such cases, we applied the samecriteria of the threshold which is set at the midpoints of thelower and higher peaks. Although other rejection methodswere tested, such as negative control +2SD or positivecontrol �2SD, they resulted in many false-positive results.Finally, we concluded that this simple method using themidpoint threshold was the most reliable and accurateaccording to a blind test with some of the fully sequencedstrains (data not shown).At the beginning of this study, we used the one-probe

one-gene array design. The accuracy of overall genotypingwas 98.2% on average (data not shown). Most of thegenotyping failures were due to false-positive results. Weanalyzed how many nucleotide differences the probes candetect. Probes containing 4 or less nucleotide mismatcheswere detected positively. Probes with eight or morenucleotide mismatches gave positive signals with a prob-ability of less than 10%. The probability of showingpositive signals with probes containing 5–8 nucleotidemismatches was in between. With such moderate strin-gency in preparation to the variance of genome sequence,occasional cross-reacting spots occurred, such as a sea spotthat cross-reacted with other types of enterotoxin genesbecause of sequence similarity. Finally, we reduced theprobability of a false-positive result by designing two or

3.5 4 4.5 5

3.5 4 4.5 5

pearance frequency of probes is presented as a function of the common

ent the results of strains, Mu50, NCTC8325,COL and N315, respectively.

nts of probes.

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more probes for each gene. By using multiple oligonucleo-tide probes to detect genes, the interference of the smallnumber of cross-reacting spots were minimized in genotypeidentification.

Finally, the microarray-based assay could accuratelydetect all 164 genes with 100% accuracy. The results ofmicroarray analysis of all 221 genes for the seven strainsare shown in Supplementary Data. The results demonstratethe high performance of the microarray in discriminatingthe genes. To evaluate the reproducibility and reliability ofhybridization signals, three independent operators werelabeled with genomic DNA of strain N315 and hybridized.Pearson’s correlation coefficients of 0.93–0.97 indicate thereproducibility of hybridization between replicate micro-arrays. The genotyping results of the three independentoperators were accurate and had 100% specificity. Thecorrelation coefficients and the genotyping results demon-strated that the microarray is a sufficiently accurate system.

3.4. Comparison of microarray results with PCR

amplification results for 18 chosen genes

To further evaluate the microarray, we studied genes thatare absent from the seven genome-sequenced strains, butwere detected in another 27 strains (Table 2). The genes werepotentially transmissible drug resistance genes, toxin genes,or several genes of phylogenetic interest. We performedPCR to confirm the absence or presence of the genes in the27 strains. Toxin genes eta, etb, etd, sec1, seb, sed, see, andsej 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, respec-tively. Enterococcus-derived drug resistance genes vanA andvanB were found in E. faecium strains 1658 and 1732,respectively (Table 2). Capsule genes capH1, capI1, andcapJ1 were carried by strain JSCS6488. We further choseanother 14 genes, such as mecA, tnpA, tnpB, tnpC, capsule-related genes, and enterotoxin genes, and a total of 32 geneswere compared in terms of DNA microarray and PCRamplification results. Amplicons were designed so that theyincluded regions of oligonucleotide probes in preparationfor genome sequencing when necessary.

The results obtained by microarray analysis agreed wellwith those by PCR analysis. We found a 99.4% con-cordance rate between them (859 of 864 possible geno-types). From the genotype data in Table 2, however, somediscrepancies were observed; five cases in total amongtnpC, capK5, capK8, and etb genes. In strain Ku, tnpC

showed positive hybridization signal but no PCR amplifi-cation signal. In strain JCSC6489, etb gave an array-positive signal although the intensity was weak and thesignal existed in the critical zone for positive judgment,whereas it was not amplified by PCR. Nonspecifichybridization may give a false-positive result because thestrains 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 caseof capK5 in strain L20A and strain JCSC6084 and capK8 in

strain JCSC4711, only weak hybridization signals weredetected and no conclusive judgment could be made. Onthe other hand, it was difficult to judge PCR amplificationsignals of capsule genes capK5 and capK8, because they didnot show strong amplification signals. The hybridizationsignals of these two genes, however, were detectedaccurately in the seven sequenced strains with sufficientsignal intensities.

3.5. Comparison of coagulase typing by microarray with

that by serological test

In the case of coagulases, we compared microarrayresults with the results of serological test. As mentionedearlier, two probes were prepared for each coagulase. Theresults are presented in Table 3. Coagulase types deter-mined with the microarray agreed with their phenotypesdetermined by the serological test. However, there weretwo strains whose coagulase types could not be judged bythe microarray-based assay. Type V coagulase of strain(serotype) JCSC2167 gave negative signal for one of thetype V probes. On the other hand, neither of the twoprobes 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 twostrains. It was found that the positive-signal probe for typeV coagulase presented only one base mismatch against thesequence of JCSC2167 coagulase, while the negative-signalprobe exhibited nine base mismatches against the corre-sponding sequence. Regarding type VI coagulase ofJCSC1469, both probes exhibited 14 base mismatchesagainst the coagulase sequence. It is reasonable thatmismatches exceeding nine bases would not give positivehybridization signals. Therefore, sequence variations thatcould not be detected by the present serological test weredetected by the microarray-based assay.Sequencing of the coagulase gene revealed that the two

strains, JCSC2167 and JCSC1469, could not be classifiedinto any known genotypes and may therefore representnovel genotypes. Notably, the sequence correspondencebetween the untypeable coagulase gene of JCSC2167 andtype V coagulase gene was found to be approximately88.3%. On the other hand, the coagulase gene sequence ofJCSC1469 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 sequencedifferences, although the phenotypes were the same.

3.6. Validation of microarray-based assignment of SCCmec

elements

The SCCmec hybridization profiles of 34 strains thatinclude strains used in the PCR verification are shown and

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Table 2

Comparison of array hybridization with PCR amplification 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 *seb *sec1 *sed *see seg seh sei *sej *eta *etb *etd

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 +/+ +/+ +/+ +/+ �/� �/� �/� �/� �/� �/� �/� �/� �/� +/+ +/+ �/� �/� �/� �/� �/� +/+ �/� �/� �/� �/� �/� �/� +/+ �/� �/� �/� �/�

**N3�15 +/+ +/+ +/+ +/+ �/� �/� �/� �/� �/� �/� �/� +/+ +/+ +/+ +/+ �/� �/� �/� �/� �/� �/� �/� �/� �/� �/� +/+ �/� +/+ �/� �/� �/� �/�

+/+: 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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compared in Table 4. Four strains previously determinedas MSSA were confirmed 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 conven-

tional typing results. Three additional findings wereobtained from the microarray analysis. Type II SCCmec

strain MRSA252 was positive for both types IIa and IVcspecific regions. To explain this result, we rechecked thefully determined E-MRSA252 genome sequence and foundthat it possessed the same sequence as type IVc specificgene (2121 bp) and percentage matching of the overlapregion was equal to 100%. Thus, the microarray correctlyshowed that the strain possesses types IIa and IVc specificregions.

Tn554 is known to be inserted only in types IIa, IIb, III,IVc, and IVd SCCmec [4]. In the microarray analysis, thefive genes related to Tn554 showed positive signals for theabove five types of strains. It was found that Tn554 genesgave also positive signals even in strains L20A andJCSC6084 that have type I SCCmec. We found concor-dance of DNA microarray data with PCR amplificationdata of the five genes of those two strains. In strain Ku, thetnpC gene was detected, although Ku is an MSSA. Aspointed out in Section 3.4, PCR gave a negative result.

4. Discussion

We have developed a microarray-based assay for thedetection and identification of staphylococcal genes. Theassay was based on random-primer labeling of the wholegenome region. We validated the assay with three differentapproaches. First, using labeled genome targets of strainN315, the microarray results were proven to be highlyreproducible when tested with three different operators atthe optimal temperature for hybridization. Second, wevalidated the assay using DNAs from seven strains whoseentire nucleotide sequences have been determined. Themicroarray showed 100% genotyping accuracy for thestrains and detected all the 164 genes present in the strains.Thus, it was confirmed that the use of multiple oligonu-cleotide probes for the detection of a staphylococcal genehad the advantages of increased specificity and sensitivity.Third, 18 genes that were absent from the seven strains butwere present in some of the tested strains were used tovalidate the assay system by comparison with PCRamplification results. For the 18 genes, PCR amplificationdata were highly consistent with microarray hybridizationdata, and there was only one case of discrepancy. Weobtained 99.5% concordance rate between the twomethods. However, for the other 14 genes tested at thesame time, which were present in the seven strains, fivecases of discrepancy were found. Using 34 strains in total,we validated 182 genes, and the total genotyping accuracyof the 182 genes was 97.8% (178/182 genes). For the genejudgments in total with 164 gene probes of the seven strains

and 32 gene probes of the 27 strains, the concordance ratewas as high as 99.8% (2007/2012 judgments).We adopted 45 mer oligonucleotide probes in the

microarray because they present two advantages. One issequence sensitivity, as the 45mer probes can detect smallsequence differences among homologous types and genefamilies. For example, the probes for coagulase typingdetected such differences. Coagulase types determined withthe microarray coincided with their phenotypes determinedby the serological test except for two strains, JCSC2167and JCSC1469. For the sequence of JCSC2167 coagulase,the probe that gave no hybridization signals exhibited ninebase mismatches against the corresponding sequence.Regarding type VI coagulase of JCSC1469, the two probesthat gave no hybridization signals possessed 14 basemismatches against the corresponding coagulase sequence.Overall, the microarray detected small sequence differencesand unexpectedly frequent variations of genome sequenceseven within a typed family of coagulase. Serological typingseems to be insensitive to small variations detectable byDNA-based methods. The other advantage of the micro-array method is that it enables us to find consensussequences within the same gene. Although such consensussequences do not enable probes to identify 4 or less basemismatches among the same gene of different strains, theycan indicate which family the strains belong to. Using sucha consensus sequence probe, we can avoid the situationobserved in JCSC1469 where all the coagulase probes gavenegative results and the existence of coagulase gene itselfcould not be determined.Five cases of discrepancy were observed between

microarray and PCR amplification results. In the case oftnpC in strain Ku, the hybridization signal was significantlystrong so that the possibility of misrecognition ormisjudgment was low. Mutations in the primer regionsmay cause failure of amplification. The hybridizationresults of tnpC in the seven genome-sequenced strains wereconsistent. Ku is an MSSA but recently, it has been pointedout that some tnpC genes exist independently of SCCmec

[4]. There also exists a gene that may be responsible for afalse-positive result. For the etb gene, nonspecific hybridi-zation may produce a false-positive result because thestrains have a homologous sequence to the oligonucleotideprobes. In such a case, only weak hybridization signals areproduced, which are found in the valley between positiveand negative hybridization signals in a histogram. In thecases of capsule genes capK5 and capK8, a possibility ofcrosstalk among PCR primers and capsule gene family canbe considered, in addition to cross hybridization in themicroarray.Collective investigation with microarray probes was also

found to be effective in the analysis of the structure ofSCCmec. Here, we followed the present criteria used forclassification: namely, the structure of the mecA complex(mecA gene with upstream regulatory region), the nature ofccr gene complexes, and the function associated with theinsertion and excision of the mec element at the specific

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Table 3

Results of array-based assignment of coagulases and their corresponding phenotypes

Phenotype starin I II III IV V V VI VII VIII IX X

JCSC3057 N315 NTCT8325 MRSA252 JCSC6306 JCSC2167 JCSC1469 MW2 Ku JCSC4711 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

determined by the array

I 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-specific probes were positive or not.

J. Otsuka et al. / Molecular and Cellular Probes 22 (2008) 1–1310

chromosomal site of orfX [36]. The microarray-basedmethod provided maximum resolution for various struc-tural variants of the SCCmec element that we haveidentified in association with various MRSA epidemiolo-gical types. This method enabled identification of not onlythe mecA complex but also the ccr gene complex: it wascapable of discriminating six major SCCmec types andsubtypes (Table 4). Several PCR-based methods areavailable 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 ofgene-specific primers or a combination of universalforward primers and specific reverse primers. One draw-back of all the present PCR-based methods is that they donot exhibit high-throughput performance. As severalseparate reactions are required in order to distinguishseveral genes, the diagnostic process is time consuming andlaborious. One problem in analyzing the relationshipbetween the presence of a specific gene (or a combinationof genes) and staphylococcal infection is that it is necessaryto characterize gene combinations of infectious strains on alarge scale. DNA microarray provides a straightforwardsolution to this problem. In one leading study, PCRproducts were used as probes [44,45]. In microarrays thatuse PCR products as probes, specific gene regions for theidentification of homologous gene families and typescannot be selected as freely as when oligonucleotide probesare used because the lengths of PCR products used as

probes usually exceed 100 bases. In addition, use of PCRproducts as probes would result in low hybridizationspecificity compared with use of oligonucleotide probesbecause their lengths would easily cause cross hybridiza-tion. As has been discussed, the use of oligonucleotideprobes instead of PCR products as probes enables us toselect specific probe regions for the identification ofhomologous gene families and types, facilitating andimproving the microarray performance, since no PCRand no template DNA of reference strains are necessary.Two kinds of oligonucleotide probe microarrays were

mentioned in the Introduction [29,30]. One was a wholegenome microarray covering approximately 90% of threeS. aureus strain genomes [29]. The other one focused on383 selected genes [30]. To design those probes, basicsequences were adopted from Mu50, N315, MW2 or COL.In contrast to single-color hybridization presented in thispaper, two-color comparative hybridization was appliedwith 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 smallnumber of genes with single-color labeling, is preferable.Due to space limitations, we briefly discussed the

genes involved in epidemiological classification, such ascoagulase and SCCmec genes. To determine the clinicalsignificance of genes encoding virulent proteins, we havestarted a separate study of clinical MRSA strains to

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Table 4

Results of array-based assignment of SCCmec elements

Strain COL L20 N315 MRSA252 JCSC3057 85/

2082

MW2 JCSC1978 MR108 JCSC4469 WIS JCSC6054 MSSA476 Ku Strain1658 Strain1732 JCSC4519

NCTC10442 JCSC6084 Mu50 85/

3907

JCSC2167 NCTC8325 JCSC4607

JCSC1469 JCSC4711 JCSC6306

JCSC4712 JCSC6488

JCSC6489

JCSC6490

JCSC6491

JCSC6492

TY114

SCCmec allotype

determined by other

studies

I Not tested II a II a II b III IV a IV b IV c IV d V VI MSSA MSSA E. faecalis E. faecalis Not tested

Array judgment I I0 II a II a0 II b III IV a IV b IV c IV d V VI No SCC No

SCC

No SCC No SCC No SCC

mecA gene complex B B A A A A B B B B mecA

only

B None None None None None

ccr complex Type 1 Type 1 Type

2

Type 2 Type 2 Type3 Type 2 Type 2 Type 2 Type 2 ccrC Type 4 None None None None None

J1 region I I II a II a and

IV c

II b III IV a IV b IV c IV d V VI None None None None None

Genes in Tn554

TnpA in Tn554 � + + + + + � � + + � � � � � � �

TnpB in Tn554 � + + + + + � � + + � � � � � � �

TnpC in Tn554 � + + + + + � � + + � � � + � � �

ermA � + + + + + � � + + � � � � � � �

ant(9)(spectinomycin) � + + + + + � � + + � � � � � � �

IS431* + + + + + + + + + + + + � � + � �

+: array hybridization signals were positive.

Bold: the bold represents the genes amplified using PCR.

*IS431 is the SCCmec specific sequence. We designed probes in this sequence as the positive control for SCCmec elements.

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correlate the repertoire of virulence genes carried by thestrains with the clinical picture of the infection caused bythem. Collective gene assay with reliable and cost-effectivemicroarrays will enable us to obtain information on thereal clinical impact of staphylococcal virulence genes.

5. Conclusion

In conclusion, the microarray-based assay described hereis a powerful tool for the analysis of S. aureus strains. Weapplied this assay to 34 standard strains and validated theresults 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 high-throughput screening and accurate genotyping of staphy-lococcal genes, which are required for the epidemiologicalstudy of S. aureus clinical strains.

Acknowledgments

This work was supported by a grant from the ‘‘Creationand Support Program for Start-ups from Universities’’ ofthe Japan Science and Technology Agency.

Appendix A. Supplementary data

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

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