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Unexpected Diversity of Staphylococcal Cassette Chromosome mec

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Unexpected Diversity of Staphylococcal Cassette Chromosome mec

     JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2010, p. 36283634 Vol. 48, No. 10 0095-1137/10/$12.00 doi:10.1128/JCM.00351-10 Copyright ? 2010, American Society for Microbiology. All Rights Reserved.

    Unexpected Diversity of Staphylococcal Cassette Chromosome mec Type IV in Methicillin-Resistant Staphylococcus aureus Strains

    12322,4Ying Liu, Fanrong Kong, Meng Xiao, Qinning Wang,Matthew OSullivan, 2,412,4Vitali Sintchenko,Lin Ma,and Gwendolyn L. Gilbert*

    1Department of Dermatology, Beijing Childrens Hospital, Capital Medical University, Beijing, Peoples Republic of China; Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research (ICPMR), 2Westmead, New South Wales, Australia; Life Science College, Peking University, Beijing, Peoples Republic of 34China; and Sydney Medical School, University of Sydney, Sydney, Australia

    Received 21 February 2010/Returned for modi(cation 6 April 2010/Accepted 23 July 2010

    Staphylococcal cassette chromosome mec (SCCmec) is a large mobile genetic element which is used fre- quently for subtyping of methicillin-resistant Staphylococcus aureus (MRSA) strains. MRSA SCCmec type IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classied as SCCmec type IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spa typing and pulsed-eld gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmec type IV sequences. A set of 131 MRSA SCCmec type IV isolates were classied into 79 subtypes by this method. There was considerable concordance between SCCmec type IV subtyping, spa typing, and PFGE patterns for clinical isolates, and the stability of SCCmec type IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpsons index of diversity of 0.975. SCCmec type IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmec type IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection.

     Methicillin-resistant Staphylococcus aureus (MRSA) strains MRSA) and can be found in coagulase-negative staphylococci, carry a large heterologous mobile genetic elementstaphylo- including community-acquired methicillin-resistant Staphylo- coccal cassette chromosome mec (SCCmec)which is inte- coccus epidermidis (C-MRSE) (9, 19, 25, 29). Perhaps as a grated at the 3 end of open reading frame X (orfX) at the consequence of its enhanced mobility, SCCmec type IV is also speci(c site attBscc, located close to the origin of replication in more variable than the other SCCmec types, and 10 subtypes the staphylococcal chromosome. SCCmec contains character- (IVa through IVj) have been reported so far (1, 13, 17, 18, 26). istic combinations of two essential genetic components that Variable targets in the MRSA SCCmec type IV J regions de(ne the SCCmec type: the mec gene complex, with mecA and have been used for subtyping in various multiplex PCR its regulator genes, and the cassette chromosome recombinase (mPCR) formats. For example, Zhang et al. (31) identi(ed (ccr) gene complex, which facilitates mobility. So far, eight subtypes IVa to IVd by using mPCR with (ve pairs of primers, SCCmec types (I to VIII) have been characterized (5, 11, 12, and Milheiric?o et al. (21) differentiated subtypes IVa to IVd, 23, 27, 30). The remaining parts of SCCmec are called J regions IVg, and IVh by employing seven pairs of primers. However, (J1, J2, and J3). Variations in the J regions (within the same the proportion of MRSA SCCmec type IV strains which are mec-ccr complex combination) are used to de(ne SCCmec nonsubtypable by existing methods remains high, reecting subtypes. their variable structure (21). MRSA SCCmec type IV is one of the most signi(cant and Our multiplex PCR-based reverse line blot (mPCR/RLB) challenging types to characterize, as it is the shortest and most hybridization assay can overcome the limitations of current mobile type (24). It not only predominates among community- methods (15). The high speci(city and sensitivity conferred by acquired MRSA (CA-MRSA) strains (6) but also is associated the use of membrane-bound sequence-speci(c probes and with several genetic lineages of hospital-acquired MRSA (HA- electrochemiluminescence to detect hybridization allow simul- taneous ampli(cation, in megaplex PCRs, of a large number * Corresponding author. Mailing address: Centre for Infectious Dis- of targets and ef(cient detection of products (15). We have eases and Microbiology (CIDM), Institute of Clinical Pathology and successfully applied this technique to molecular typing of sev- Medical Research (ICPMR), Westmead Hospital, Darcy Road, West- eral bacterial pathogens, including Streptococcus agalactiae mead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: lyn.gilbert@sydney.edu.au. (16), Streptococcus pneumoniae (14), and Staphylococcus au- Supplemental material for this article may be found at http://jcm reus (3). The format used in this study involves ampli(cation of .asm.org/. more than 60 targets in two mPCRs, with corresponding tar- Y.L., F.K., and M.X. contributed equally to this work. get-speci(c probes distributed between two reusable mem- Published ahead of print on 4 August 2010.

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FIG. 1. RLB patterns of 40 MRSA reference strains. The two membranes are aligned side by side and show 32 and 33 probes, including one

    control probe (mecASP) (for a total of 63 subtype-speci(c probes). Rows: P, positive control; N, negative control; 1, PAH1; 2, E804531; 3, F829549; 4, MW2; 5, 13792-4492; 6, IP01M1081; 7, WA01; 8, WA02; 9, FH43; 10, SJOG 30; 11, RHH58; 12, RPH85; 13, B8-10; 14, CH 16; 15, WA08; 16, RHH10; 17, WA15; 18, WA17; 19, WA19; 20, WA20; 21, CH97; 22, DEN2988; 23, WA13; 24, WA23; 25, PC8; 26, IP01M2046; 27, WA24; 28, WA26; 29, WA29; 30, WA30; 31, WA31; 32, WA33; 33, WA37; 34, WA39; 35, WA41; 36, WA42; 37, RBH98; 38, WA47; 39, WA48; 40, WA54.

    collected at intervals of 1 to 30 months (median, 5 months), were used to assess branes: up to 43 isolates can be tested simultaneously on each in vivo stability. membrane pair. Details of the reference strains and clinical isolates are provided in Fig. 2 to 4. The aim of this study was to map MRSA SCCmec type IV Isolates were stored at 70?C until DNA extracts were prepared as previously open reading frames (ORFs) by using a comprehensive mPCR/ described (3). These MRSA strains were all shown to possess SCCmec type IV RLB assay and to explore the diversity of SCCmec type IV by a previously described method (2, 21, 31). Primer and probe design for mPCR/RLB assay and in silico experiments. genotypes of importance for infection control. Primers were designed to amplify each of the known ORFs of SCCmec type IV, based on the sequences of six type IV subtypes available in GenBank (http://www MATERIALS AND METHODS .ncbi.nlm.nih.gov) (accession numbers AB063172 [SCCmec type IVa], AB063173 [SCCmec type IVb], AB096217 [SCCmec type IVc], AB097677 [SCCmec type Bacterial isolates. (i) Reference strains. Fifty-two well-characterized MRSA IVd], DQ106887 [SCCmec type IVg], and AF411936 [SCCmec type IVh]). One reference strains from previously described collections (3, 4, 7), (ve strains primer from each pair was labeled with biotin. Immediate downstream or up- with published whole-genome sequences not containing SCCmec type IV, and stream sequences were used to design antisense or sense probes for mPCR/RLB one strain with a published whole-genome sequence containing SCCmec type assay as described previously (15). Some primers were used to amplify more than IV (MW2) were used in this study. The six sequenced strains and their one target sequence due to homology between ORFs. Two probes for ORF R009 corresponding whole-genome sequence accession numbers were as follows: were used because of its sequence diversity; other ORFs were detected with one COL (SCCmec type I; GenBank accession number CP000046), MU3 (SCCmec type probe. In all, 62 primer pairs were utilized in two mPCRs, with product detection II; GenBank accession number AP009324), MU50 (SCCmec type II; GenBank relying on 63 probes distributed across two RLB membranes (see Table S1 in the accession number BA000017), NCTC8325 (S. aureus; GenBank accession num- supplemental material). In silico experiments were conducted with these primers ber CP000253), ATCC 12228 (S. epidermidis; GenBank accession number and probes to ensure oligonucleotide speci(city, using 21 SCCmec region se- AE015929), and MW2 (SCCmec type IV; GenBank accession number quences published in NCBI GenBank, including all 16 SCCmec type IV se- BA000033). quences that were of adequate length and from MRSA strains. The GenBank (ii) Clinical isolates. Forty-one unique clinical isolates, collected from differ- accession numbers of these sequences were as follows: AB063172, EF596937, ent patients in three tertiary hospitals in Sydney, Australia, were used. In addi- tion, another 38 paired isolates from 19 MRSA-colonized or -infected patients, AB266531, EU437549, and EU437550 for SCCmec type IVa; AB063173 for

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     VOL. 48, 2010 HIGH DIVERSITY OF MRSA SCCmec TYPE IV 3631

    SCCmec type IVb; AB266532, AB096217, and AY271717 for SCCmec type IVc; RESULTS AND DISCUSSION AB097677 for SCCmec type IVd; AJ810121 for SCCmec type IVe; DQ106887 for SCCmec type IV subtyping by mPCR/RLB assay. The RLB SCCmec type IVg; AF411936 for SCCmec type IVh; AB425824 for SCCmec type IVj; CP000730 for SCCmec type IV USA300_TCH1516; CP000253 for methi- patterns on both membranes are shown in Fig. 1 for 40 of the cillin-susceptible S. aureus (MSSA) NCTC8325 whole-genome sequence; 52 MRSA reference strains. Figure 2 shows the relationships BA000033 for SCCmec type IV; AE015929 for methicillin-susceptible Staphylo- between mPCR/RLB results for the MRSA SCCmec type IV coccus epidermidis ATCC 12228 whole-genome sequence; AP009324 for SCCmec reference strains (labeled R) and those of in silico alignments type II; BA000017 for SCCmec type II; and CP000255 for SCCmec type I. GenBank sequence searches and alignments and design of primers and probes of primers and probes against the sequences of 16 SCCmec were conducted using BioManager (Sydney Bioinformatics [http://biomanager type IV strains from GenBank (labeled G). Overall, 41 distinct .info/]) and Sigma DNA calculator browsers (Sigma-Genosys). subtypes were identi(ed among the 52 reference strains. Clus- uPCR and mPCR. PCR ampli(cations were performed in a thermal cycler ter analysis revealed that isolates with the same SCCmec sub- with HotStar Taq DNA polymerase (Qiagen, Valencia, CA). A 25- l uniplex PCR (uPCR) mixture was prepared as follows: 2.5 l of 10 PCR buffer with a type, as de(ned by Milheiric?o et al. (21), did not always group (nal MgCl2 concentration of 1.5 mM, a 0.2 mM concentration of each de- together and that none of the 52 reference strains corre- oxynucleoside triphosphate (dNTP), 12.5 M (each) forward and reverse prim- sponded exactly with the six type strains, suggesting that exist- ers, 2 l template DNA ( 43 g/ml; equivalent to (ve MRSA colonies), 0.2 l ing SCCmec type IV subtyping methods may not be able to Qiagen Hotstar Taq polymerase (5 U/ l), and molecular biology-grade HO 2(Eppendorf) were added to a total volume of 25 l. represent complex phylogenetic relationships for this diverse Two mPCR mixes were prepared: the (rst contained 32 primer pairs, and the MRSA type. Weak signals were noted for some probes with a second contained 31 primer pairs. Each mPCR mix consisted of 2 l of DNA few isolates, which were likely due to minor sequence varia- template and 3 l of 10 PCR buffer with 1.5 mM ((nal concentration) MgCl 2tions in the probe regions, as reported previously (28). (Qiagen), a 0.2 mM concentration of each dNTP, 1.5 U HotStar Taq DNA The mPCR/RLB assay classi(ed 79 clinical isolates into 38 polymerase, and 12.5 M (each) forward and reverse primers. Molecular biol- ogy-grade HO (Eppendorf) was added to a total volume of 30 l. PCR was 2subtypes (Fig. 3). Isolates with similar spa types were clustered performed according to the Qiagen HotStar Taq polymerase kit instructions, as together, suggesting that they have epidemiologic relation- follows: 95?C for 15 min; 35 cycles of 94?C for 30 s, 55?C for 30 s, and 72?C for ships. In contrast, there was a poor correlation between 45 s; and 72?C for 10 min. PCR products were stained with SYBR Safe DNA gel mPCR/RLB clusters and spa types of the reference strains, stain and visualized in a 2% agarose gel. RLB hybridization. The RLB hybridization assay was performed as described which are epidemiologically unrelated. previously (3, 15), using two membranes. The hybridization temperature was In the study of in vivo stability, members of 12 of 19 isolate 60?C, the washed membrane was incubated in peroxidase-labeled streptavidin pairs produced identical results by PFGE, mPCR/RLB assay, conjugate (Roche, Mannheim, Germany) at 42?C for 60 min, and the time of and spa typing. The members of seven pairs differed from each exposure to X-ray (lm (Hyper(lm; Amersham) was 5 min. RLB results were regarded as positive when a hybridization dot signal was clearly visible. Addi- other by at least one band in PFGE gels (data not shown), but tional uPCR testing was conducted to resolve any weak signals produced by a test among these, (ve pairs had identical mPCR/RLB and spa isolate and an individual probe. Weak mPCR/RLB results con(rmed by the typing results between their members. Assuming that the positive uPCR were treated as positive results. members of these pairs represented essentially the same Quality control of mPCR/RLB results. To ensure the reproducibility of mPCR/RLB results, positive and negative controls were run on each membrane. strains, carried for various periods by the same patients, these Positive controls were constructed by mixing extracted DNAs from selected results indicate that the SCCmec type IV loci remain stable for strains to produce a sample expected to be positive for each probe on the at least several months. They imply that despite its high dis- membrane, with the exception of the CM001SP, CM002SP, and PK01SP probes, criminatory power, the mPCR/RLB typing method is more for which no strains in our collection were positive. The negative (no DNA) stable than PFGE over time for the same or epidemiologically control was master mix only. A signal produced by the negative control was assumed to be due to contamination, and the assay was repeated in such a case. related strains, which is important in the investigation of pro- PFGE. Pulsed-(eld gel electrophoresis (PFGE) analysis of SmaI-digested longed outbreaks. Two isolate pairs had different mPCR/RLB DNA was performed using the protocol described by McDougal et al. (20). Gels patterns between members (22a-22b and 56a-56b) (Fig. 3); were stained with ethidium bromide and photographed under UV light with a isolates 22a and 22b also had different spa types. The major charge-coupled device (CCD) camera. PFGE patterns were analyzed and com- pared using BioNumerics software (version 4.61; Applied Maths). The Dice differences in mPCR/RLB patterns between the members of coef(cient was used for pairwise comparisons of patterns, and the unweighted- these two pairs suggest that the patients original colonizing pair group method using average linkages (UPGMA) was used for pattern strains were replaced by different ones. This was supported by groupings. Position tolerance and optimization were both set at 1%. the PFGE results for these pairs, which showed eight and three spa typing. spa typing was performed as previously described (3), and types were assigned by consulting the Ridom SpaServer (8; http://spaserver.ridom.de). band differences between 22a and 22b and between 56a and Statistical analysis. The mPCR/RLB results were recorded as binary data and 56b, respectively. exported into BioNumerics software (version 4.61; Applied Maths). Dendro- A total of 79 SCCmec type IV subtypes in the whole set of grams were generated using the categorical coef(cient and clustering by the 131 isolates were identi(ed using mPCR/RLB assay (with 63 UPGMA algorithm. Selection of discriminatory targets was performed using the probes and targets), with a Simpsons index of diversity (D) of AuSeTTS program (http://www.cidmpublichealth.org/pages/ausetts.html).

    FIG. 2. Dendrogram showing in vitro mPCR/RLB results for 52 MRSA reference strains (labeled class R), aligned with simulated in silico mPCR/RLB results for 16 SCCmec subtype IV gene cluster sequences published in GenBank (labeled class G), based on BLASTn searches against mPCR/RLB probe sequences. For the GenBank SCCmec sequences (class G), black squares represent perfect matches ( 99.9% identity between sequences) with probe sequences and gray squares represent probes for which there was no matching GenBank sequence. For the 52 MRSA reference strains (class R), the black and gray squares represent positive and negative signals, respectively, on the RLB membrane. Abbreviations for geographic sources of Australian reference strains: WA, Western Australia; NSW, New South Wales; TAS, Tasmania; QLD, Queensland.

FIG. 3. Dendrogram showing mPCR/RLB results for 79 MRSA clinical isolates. The black and gray squares represent positive and negative

signals, respectively, on the RLB membrane. Isolates labeled with the same number and either a or b represent paired isolates from the same patient. Seventeen of 19 pairs had the same mPCR/RLB and spa typing results; for the 56a-56b pair, mPCR/RLB pro(les were different but spa types were the same between its members, and for the 22a-22b pair, both mPCR/RLB and spa typing results were different (suggesting that the patients colonizing strains had changed over time). Abbreviations for sources (three public hospitals in metropolitan Sydney, Australia): A, Westmead; B, North Shore; and C, Ryde.

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FIG. 4. Comparison of in silico (G) and in vitro (I) results for (ve isolates with published whole-genome sequences that do not contain SCCmec

    type IV and one that contains SCCmec type IV. The reference strains and corresponding GenBank accession numbers are as follows: rows 1 and 2, SCCmec type I strain COL (class I) and GenBank accession number CP000046 (class G); rows 3 and 4, SCCmec type IV GenBank accession number BA000033 (class G) and strain MW2 (class I); rows 5 to 8, SCCmec type II strains MU3 (class I) and MU50 (class I) and GenBank accession numbers AP009324 (class I) and BA000017 (class G); rows 9 and 10, methicillin-susceptible S. aureus GenBank accession number CP000253 (class G) and strain NCTC8325 (class I); and rows 11 and 12, S. epidermidis GenBank accession number AE015929 (class G) and strain ATCC 12228 (class I). For GenBank SCCmec sequences (class G), the black squares represent perfect matches ( 99.9% identity between sequences) of probe sequences with the GenBank SCCmec sequences, and the gray squares represent probes for which there were no matching GenBank sequences. For the six reference strain isolates (class I), the black and gray squares represent positive and negative signals, respectively, on the RLB membrane.

    0.975. Using an in-house computer program, AuSeTTS (auto- Relative efciency of mPCR/RLB assay. In contrast to tra- mated selection of typing target subsets [available at http: ditional approaches to SCCmec subtyping based on gel elec- //www.cidmpublichealth.org/]), we determined that using a trophoresis, the use of the mPCR/RLB assay enables the si-

    subset of only 20 probes could achieve the same level of dis- multaneous screening of up to 43 isolates, using two multiplex crimination as the whole (see Table S1 in the supplemental PCRs with a short turnaround time. Culture of isolates, DNA material). extraction, mPCR setup and running time, and RLB hybrid- Comparison of in vitro and in silico analyses. Results of the ization require no more than two working days. The prepara- in vitro mPCR/RLB assay for six reference strains and of in tion of the RLB membrane takes less than 2 h, and the mem- silico analysis of their corresponding whole-genome sequences brane can be reused at least 20 times, reducing the cost of against mPCR/RLB probes are shown in Fig. 4. As predicted consumables to approximately AU$10 per isolate. The mPCR/ by in silico analysis of whole-genome sequences, a number of RLB assay can easily be transferred to other laboratories, and probes hybridized with (ve strains that do not contain SCCmec the results are represented as binary data, which can be shared type IV as well as with the one SCCmec type IV-containing between laboratories. The reproducibility of the mPCR/RLB strain (10, 22). This was most apparent for SCCmec type II assay between laboratories should be excellent, provided that strains MU3 and MU50, since SCCmec type II has consider- the same primers, probes, reagents, PCR conditions, and pos- able homology with SCCmec type IV, as shown by positive itive control are used (15). Contamination between samples is results with the IVhSP1, IVhSP2, IVhSP3, IVhSP4, IVSP5, rare and is easily avoided by careful technique and the use of PK02SP, and aacA-aphDSP probes. No corresponding se- controls. The method is as discriminatory as other MRSA quences were found for probes PK02SP and aacA-aphDSP in typing methods and is stable over time. the whole-genome sequences of these two SCCmec type II In combination, these features make mPCR/RLB assays strains, suggesting that the reference strain cultures tested had very suitable for rapid epidemiological studies of large num- acquired mobile (e.g., phage or plasmid) DNA, possibly as a bers of MRSA SCCmec type IV isolates for investigations of result of subculture. Three of the cross-reacting targets (aacA- potential outbreaks. The MRSA SCCmec type IV genomic aphD-IV3, IVhSP1, and IVhSP3) were in the subset of 20 region appears to be more variable than previously thought, which contributed to the discriminatory power of the assay, but but our method discriminates among currently recognized ma- the other 4 could be omitted from the (nal assay con(guration jor SCCmec type IV subtypes and can easily be modi(ed to without a loss of discrimination. The cross-reactions are un- accommodate probes for newly identi(ed SCCmec elements. likely to be confusing in practice, since the system is designed

    Selective sequencing of the 20 most discriminatory markers for rapid subtyping of SCCmec type IV strains rather than

    could allow development of an even more accurate and repro- identi(cation of SCCmec types.

3634 LIU ET AL. J. CLIN. MICROBIOL.

2004. Novel type V staphylococcal cassette chromosome mec driven by a ducible sequence-based typing system, which would be appli- novel cassette chromosome recombinase, ccrC. Antimicrob. Agents Che- cable to testing of small numbers of isolates. mother. 48:26372651. In conclusion, we have shown that SCCmec type IV has a 13. Ito, T., K. Okuma, X. X. Ma, H. Yuzawa, and K. Hiramatsu. 2003. Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: much higher level of diversity than previously described. The genomic island SCC. Drug Resist. Updat. 6:4152. mPCR/RLB-based SCCmec typing system improves our capac- 14. Kong, F., M. Brown, A. Sabananthan, X. Zeng, and G. L. Gilbert. 2006. ity to monitor the molecular evolution and spread of MRSA Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes. J. Clin. Micro- SCCmec type IV strains and contributes to effective strategies biol. 44:18871891. for infection control. The application of the mPCR/RLB hy- 15. Kong, F., and G. L. Gilbert. 2006. Multiplex PCR-based reverse line blot bridization assay to MRSA SCCmec typing enhances the spec- hybridization assay (mPCR/RLB)a practical epidemiological and diagnos- tic tool. Nat. Protoc. 1:26682680. i(city, discriminatory power, and throughput of the typing pro- 16. Kong, F., L. Ma, and G. L. Gilbert. 2005. Simultaneous detection and cedure. The simultaneous detection of up to 43 mPCR serotype identi(cation of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization. J. Med. Microbiol. 54:11331138. products in a single hybridization assay makes this assay a 17. Kwon, N. H., K. T. Park, J. S. Moon, W. K. Jung, S. H. Kim, J. M. Kim, S. K. practicable tool for rapid infection control surveillance and Hong, H. C. Koo, Y. S. Joo, and Y. H. Park. 2005. Staphylococcal cassette MRSA outbreak detection. chromosome mec (SCCmec) characterization and molecular analysis for methicillin-resistant Staphylococcus aureus and novel SCCmec subtype IVg isolated from bovine milk in Korea. J. Antimicrob. Chemother. 56:624632. ACKNOWLEDGMENTS 18. Ma, X. X., T. Ito, C. Tiensasitorn, M. Jamklang, P. Chongtrakool, S. Boyle- Vavra, R. S. Daum, and K. Hiramatsu. 2002. Novel type of staphylococcal We thank Herminia de Lencastre (Instituto de Tecnologia Química cassette chromosome mec identi(ed in community-acquired methicillin-re- e Biolo?gica [ITQB], Universidade Nova de Lisboa [UNL], Oeiras, sistant Staphylococcus aureus strains. Antimicrob. Agents Chemother. 46: Portugal), Graeme Nimmo (Queensland Health Pathology Services, 11471152. 19. Maree, C. L., R. S. Daum, S. Boyle-Vavra, K. Matayoshi, and L. G. Miller. Princess Alexandra Hospital, Brisbane, Queensland, Australia), Philip 2007. Community-associated methicillin-resistant Staphylococcus aureus iso- Giffard (Menzies School of Medical Research, Darwin, Australia), and lates causing healthcare-associated infections. Emerg. Infect. Dis. 13:236 Geoffrey Coombs (PathWest Laboratory Medicine, Royal Perth Hos- 242. pital, Perth, Western Australia) for providing the reference strains 20. McDougal, L. K., C. D. Steward, G. E. Killgore, J. M. Chaitram, S. K. used in this study. McAllister, and F. C. Tenover. 2003. Pulsed-(eld gel electrophoresis typing This work was supported partly by the Beijing Natural Science Foun- of oxacillin-resistant Staphylococcus aureus isolates from the United States: dation (grants 7062023 and 7092031) for Ying Lius visit to the Centre establishing a national database. J. Clin. Microbiol. 41:51135120. for Infectious Diseases and Microbiology. 21. Milheiric?o, C., D. C. Oliveira, and L. H. de Lancastre. 2007. 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