Validation of binary typing for Staphylococcus aureus strains

Most of the DNA-based methods for genetic typing of Staphylococcus aureus s trains generate complex banding patterns, Therefore, we have developed a bi nary typing procedure involving strain-differentiating DNA probes which wer e generated on the basis of randomly amplified polymorphic DNA (RAPD) analy sis, We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376 S. aureus strains of wh ich 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns ge nerated, 66 were selected which identified 124 unique DNA fragments, From t hese amplicons, only 12% turned out to be useful for isolate-specific binar y typing. The nature of the RAPD-generated DNA fragments Has investigated b y partial DNA sequence analysis. Several homologies with known S. aureus se quences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of S, aureus NCTC 8325 were determined by hybridization, Seven fragments were randomly dispersed along the genome , five were clustered within the 2500- to 2600-kb position of the genome, a nd the remaining four did not recognize complementary sequences in S, aureu s NCTC 8325, A total of 103 S, aureus strains (69% MRSA) were used for the validation of the binary typing technique, The 15 DNA probes provided stabl e epidemiological markers, both in vitro (type consistency after serial pas sages on culture media) and in vivo (comparison of sequential isolates reco vered from cases of persistent colonization). The discriminatory power of b inary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by ana lyzing multiple strains belonging to a multitude of different epidemiologic al clusters, was comparable to that of other genotyping techniques used, Co ntribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of S. aureus isolates.