MOLECULAR TYPING OF STAPHYLOCOCCUS-AUREUS BASED ON PCR RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM AND DNA-SEQUENCE ANALYSIS OF THE COAGULASE GENE

A typing procedure for Staphylococcus aureus was developed based on im proved PCR amplification of the coagulase gene and restriction fragmen t length polymorphism (RFLP) analysis of the product. All coagulase-po sitive staphylococci produced a single PCR amplification product of ei ther 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-r esistant 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 fou nd among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus (MSSA) exa mined. 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 se en with a heterogeneous collection of strains, together with drug-resi stant forms of S. aureus isolated in Europe and four propagating strai ns used for the international phage set. RFLP pattern 6 was given by t he Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (iso late 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8, Eight strains of MSSA gave patterns similar to those of methicilli n-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 coag ulase gene PCR products for 24 isolates of MRSA and two isolates of MS SA were sequenced for both strands. The sequences were aligned, and ev olutionary lineages were inferred based on pairwise distances between isolates.