Objectives
Staphylococcus aureus is a major human pathogen. Both its population structure and transmission patterns are complex and rapidly changing, with the emergence in the last decade of multiple community acquired MRSA (caMRSA) clones being a particularly remarkable phenomenon. Consequently there is strong justification for ongoing surveillance of this species, and this has provided the impetus for considerable activity in the development of S. aureus typing methods. Major drivers are cost, time, portability of data, discriminatory power and concordance between the type and the S. aureus population structure. The objective of this presentation is to review rapid typing methods that are designed to provide a result quickly.
Methods
The principal approaches to rapid S. aureus typing are those based on electrophoresis-based size determination of variable number tandem repeat (VNTR) loci, and those based on real-time PCR-based interrogation of single nucleotide polymorphisms (SNPs) and/or binary genes and/or VNTRs.
Results
Methods based upon multiple VNTR loci provide high discrimination, although the likely homoplastic nature of these loci complicates interpretation. Also, genotyping VNTR loci solely on basis of size can result in sequence differences being missed, and gel patterns can be difficult to digitise effectively. Extant VNTR-based methods are inherently two step because of the requirement for electrophoresis, and this impacts on the time required. Real-time PCR based methods have the advantage of being single step and closed tube, and can interrogate different classes of polymorphism. This facilitates a hierarchical approach that makes use of markers that evolve at different rates. For example, it is possible to identify SNPs that resolve the major clonal complexes, and then add resolution by determining the presence/absence of mobile genetic elements, and/or interrogating VNTRs. High resolution melt (HRM) is emerging as a convenient, robust and easily optimised technology that can facilitate the use of real-time PCR to interrogate different classes of polymorphisms, including VNTRs.
Conclusions
There is not one method suitable for all typing requirements. However, an hierarchical procedure that makes use of HRM is an example of a potentially widely applicable approach.