Rapid cost-effective subtyping of meticillin-resistant Staphylococcus aureus by denaturing HPLC

Abstract

The importance of meticillin-resistant Staphylococcus aureus (MRSA) in hospital-acquired infection is widely acknowledged. The UK government has stated that MRSA bloodstream infection rates will have to be halved by 2008. Such radical improvements will require advances on several fronts. Screening for MRSA in high-risk patients on arrival at hospital allows isolation of carriers and reduces transmission to staff and other patients. Concurrent subtyping of MRSA could also inform outbreak investigations and long-term epidemiological studies. The variability within the staphylococcal protein A, or spaA, gene-repeat region can be used as a marker of short- and long-term genetic variation. A novel application is described of denaturing HPLC (DHPLC) for rapid, inexpensive characterization of spaA gene amplification products, without the need for DNA sequence determination. The method allowed rapid and precise sizing of spaA gene-repeat regions from 99 S. aureus strains and was combined with heteroduplex analysis, using reference PCR products, to indicate the spa type of the test isolate. The method allowed subtyping of strains in less than 5 h from receipt of a primary isolation plate. When applied to an outbreak that occurred during this study, the authors were able to demonstrate relatedness of the isolates more than 5 days before results were received from a reference laboratory. If combined with direct amplification from swabs, DHPLC analysis of spaA gene variation could prove extremely valuable in outbreak investigation and MRSA surveillance.