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

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 microand 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.Strain typing techniques for Staphylococcus aureus, the leading cause of nosocomial infections (28), have become widely used. These techniques aid in both local/short-term epidemiologic outbreak investigations and global/long-term population-based studies of methicillin-resistant and -susceptible S. aureus (MRSA and MSSA, respectively). Macrorestriction digests using pulsed-field gel electrophoresis (PFGE) have been shown to be highly effective in outbreak settings (48). Multilocus enzyme electrophoresis (MLEE), multilocus sequence typing (MLST), and sequence analysis of the repeat region within the coagulase gene, i.e., coa typing, are effective techniques for analyzing S. aureus strains in long-term study settings (10,42), and the sequence analysis of the repeat region within the protein A gene, spa typing, can effectively be used in both settings (22,41)....

Assignment of Staphylococcus Isolates to Groups by spa Typing, SmaI Macrorestriction Analysis, and Multilocus Sequence Typing

“…Besides t002, which had already been reported for MRSA isolates of lineage ST5 from Central Europe (13), two other spa types have been detected (t045 and t178). Type t002 was also found in MRSA isolates of ST5 from the United States (15,16) and in the majority of MRSA isolates of these lineages from Japan and southern Korea. In this study, seven "subtypes" due to deletion, insertion, and point mutations were reported (32).…”

“…An interpretation in the sense of a direct common ancestral origin of both pathotypes of this clonal lineage would be rather speculative, since spa type t021 could have been derived from other spa types by loss of one or more final repeats of r24 (e.g., from type t012 or t018). MSSA isolates of MLST CC30 exhibiting spa types t012, t018, and t021 from the United States and from Poland have also been described previously (15,16,18).…”

“…This first became evident by the application of multilocus sequence typing (MLST) to MRSA (4, 5). At present, however, MLST is not suitable for routine infection control due to high cost, labor intensity, and lack of broad access to high-throughput DNA sequencing.Several S. aureus typing schemes targeting polymorphic DNA repeat regions in genes for microbial surface components recognizing adhesive matrix molecules have been described previously (7,9,16,27,30). They also include typing methods based on the length polymorphism in spa amplimers (9) or, more recently, on polymorphisms in multiple fragments amplified in a multiplex PCR approach for variable-number tandem repeats (7, 27).…”

“…Several S. aureus typing schemes targeting polymorphic DNA repeat regions in genes for microbial surface components recognizing adhesive matrix molecules have been described previously (7,9,16,27,30). They also include typing methods based on the length polymorphism in spa amplimers (9) or, more recently, on polymorphisms in multiple fragments amplified in a multiplex PCR approach for variable-number tandem repeats (7,27).…”

Multiplexed Genotyping of Methicillin-Resistant Staphylococcus aureus Isolates by Use of Padlock Probes and Tag Microarrays

“…The limited discriminatory power of MLST and the high costs associated with sequence analyses of seven slowly evolving genetic loci have driven the development of a number of sequence-based typing approaches that rely on the investigation of single highly variable genomic regions (5,10,19,35). Among these, the most frequently applied method explores a polymorphic repetitive region in the staphylococcal protein A (spa) gene (1,7,40).…”