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: (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 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.