Validation of binary typing for Staphylococcus aureus strains

Abstract

Most of the DNA-based methods for genetic typing of Staphylococcus aureus strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. 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 which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known S. aureus sequences 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, and the remaining four did not recognize complementary sequences in S. aureus 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 stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution 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.

FIG. 1

Physical mapping of the DNA probes on S. aureus NCTC 8325 (29) restriction fragments. (A) PFGE macrorestriction patterns of S. aureus NCTC 8325 digested with SmaI, SgrAI, and AscI (lanes 1, 2, and 3, respectively). Restriction fragments are coded by descending molecular size. (B) Example of hybridization results with probe AW-15 to PFGE patterns of S. aureus NCTC 8325. Lanes are the same as for panel A. (C) Hybridization results of probe AW-15 depicted on the physical map of S. aureus NCTC 8325. (D) Mapping results of the 15 strain-specific DNA probes (AW-1 through AW-15) to the macro-restriction fragments of the S. aureus NCTC 8325 genome. NH, no hybridization of the strain-specific DNA probe to the macrorestriction fragments.

FIG. 2

Representative example of binary typing results obtained with the complete panel of strain-specific DNA probes (AW-1 through AW-15) from three MRSA clusters VIII, IX, and X (n = 15, collection 10). Each cluster encompassed five strains (a, b, c, d, and e) and displayed identical hybridization results.

FIG. 3

(a) Dendrograms displaying the grouping of 40 unique S. aureus strains (Table 1, collections 6 and 7) on the basis of hybridization scores after binary typing with all DNA probes (dendrogram 1); deletion of probe AW-4 (dendrogram 2); deletion of probes AW-4 and AW-3 (dendrogram 3); deletion of probes AW-4, AW-3, and AW-15 (dendrogram 4); and deletion of probes AW-4, AW-3, AW-15, and AW-6 (dendrogram 5). (b) Dendrograms presenting the similarity percentages of the hybridization patterns of 10 outbreak clusters of S. aureus strains (Table 1, collection 10) obtained with the complete panel of DNA probes comprising the binary typing system (dendrogram 1); after deletion of probe AW-4 (dendrogram 2); after deletion of probes AW-4 and AW-3 (dendrogram 3); after deletion of probes AW-4, AW-3, and AW-15 (dendrogram 4); and after deletion of probes AW-4, AW-3, AW-15, and AW-6 (dendrogram 5).