Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene

Abstract

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested with AluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus (MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.