Rapid genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolates using miniaturised oligonucleotide arrays

Abstract

This study evaluated a DNA oligonucleotide array that recognised 38 different Staphylococcus aureus targets, including all relevant resistance determinants and some toxins and species-specific controls. A new method for labelling sample DNA, based on a linear multiplex amplification that incorporated biotin-labelled dUTP into the amplicon, was established, and allowed detection of hybridisation of the amplicons to the array with an enzymic precipitation reaction. The whole assay was validated by hybridisations with a panel of reference strains and cloned specific PCR products of all targets. To evaluate performance under routine conditions, the assay was used to test 100 methicillin-resistant S. aureus (MRSA) isolates collected from a university hospital in Saxony, Germany. The results showed a high correlation with conventional susceptibility data. The ermA and ermC macrolide resistance genes were found in 40% and 32% of the isolates, respectively. The most prevalent aminoglycoside resistance gene was aphA3 (57% of the isolates), followed by aacA-aphD (29%) and aadD (29%); tet genes, mupR and dfrA were rare. There were no isolates with van genes or genes involved in resistance to quinupristin-dalfopristin. Enterotoxins were detected in 27% of the isolates. Genes encoding Panton-Valentine leukocidin, toxic shock syndrome toxin and exfoliative toxins were not found. The DNA array facilitated rapid and reliable detection of resistance determinants and toxins under conditions used in a routine laboratory and has the potential to be used for array-based high-throughput screening.