Comparative Sequencing of the Serine-Aspartate Repeat-Encoding Region of the Clumping Factor B Gene ( clfB ) for Resolution within Clonal Groups of Staphylococcus aureus

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Abstract
Molecular techniques such as spa typing and multilocus sequence typing use DNA sequence data for differentiating Staphylococcus aureus isolates. Although spa typing is capable of detecting both genetic micro- and macrovariation, it has less discriminatory power than the more labor-intensive pulsed-field gel electrophoresis (PFGE) and costly genomic DNA microarray analyses. This limitation hinders strain interrogation for newly emerging clones and outbreak investigations in hospital or community settings where robust clones are endemic. To overcome this constraint, we developed a typing system using DNA sequence analysis of the serine-aspartate (SD) repeat-encoding region within the gene encoding the keratin- and fibrinogen-binding clumping factor B ( clfB typing) and tested whether it is capable of discriminating within clonal groups. We analyzed 116 S. aureus strains, and the repeat region was present in all isolates, varying in sequence and in length from 420 to 804 bp. In a sample of 36 well-characterized genetically diverse isolates, clfB typing subdivided identical spa and PFGE clusters which had been discriminated by whole-genome DNA microarray mapping. The combination of spa typing and clfB typing resulted in a discriminatory power (99.5%) substantially higher than that of spa typing alone and closely approached that of the whole-genome microarray (100.0%). clfB typing also successfully resolved genetic differences among isolates differentiated by PFGE that had been collected over short periods of time from single hospitals and that belonged to the most prevalent S. aureus clone in the United States. clfB typing demonstrated in vivo, in vitro, and interpatient transmission stability yet revealed that this locus may be recombinogenic in a primarily clonal population structure. Taken together, these data show that the SD repeat-encoding region of clfB is a highly stable marker of microvariation, that in conjunction with spa typing it may serve as a DNA sequence-based alternative to PFGE for investigating genetically similar strains, and that it is useful for analyzing collections of isolates in both long-term population-based and local epidemiologic studies.

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