A comprehensive, novel Staphylococcal Cassette Chromosome mec (SCCmec) typing assay using multiplex PCR based reverse line blot hybridization (mPCR/RLB)

  • Dr Lin Cai, Australia
  • Dr Fanrong Kong, Centre for Infectious Diseases and Microbiology, Westmead Hospital, Sydney and Western Clinical School, University of Sydney, Australia
  • Dr Qinning Wang, Australia
  • Dr Huiping Wang, Australia
  • Ms Kathryn Shaw, Australia
  • Gwendolyn Gilbert, Centre for Infectious Diseases and Microbiology, Australia

    • Objective: Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital- and community-acquired pathogen. Molecular typing can provide information about virulence, identify transmission and guide infection control. Staphylococcal cassette chromosome mec (SCCmec), carrying methicillin resistance gene, mecA, is a potential target for typing. Six SCCmec types (I-VI) and some subtypes have been characterized. Our aim was to develop a comprehensive, practical SCCmec (sub)typing system, using a multiplex PCR-based reverse line blot hybridization (mPCR/RLB) assay.
    Methods: We used 45 primer pairs, in a single mPCR, and 49 probes to study 42 previously well-characterized, MRSA reference strains, representing five SCCmec types, nine clonal clusters and 22 spa types. We compared sequences identified in the reference strains with 15 published SCCmec sequences (including all known types and subtypes) and 12 whole S. aureus genomes.
    Results: We identified 5 SCCmec types and 26 subtypes, including one of SCCmec type I (1 strain), two subtypes of SCCmecII (2 strains), eight of SCCmecIII (11 strains), eight of SCCmecIV (21 strains) and three of SCCmecV (3 strains). Four strains belonged to previously unknown types, which were most closely related to SCCmecIV and V, of which they may be new subtypes.
    Conclusions: Based on comparison of results for this small, but diverse strain collection with GenBank SCCmec sequence data, this typing scheme could potentially identify novel SCCmec types/subtypes among larger collections. It allows rapid characterisation of several SCCmec targets, with high specificity and discriminatory power and will be suitable for studies of epidemiology and genetic diversity of MRSA.