Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus

Abstract

Sequence-based epidemiological typing of methicillin-resistant Staphylococcus aureus (MRSA) has recently been promoted because it results in unambiguous data sets that can be organized in local and global databases. The replacement of previous typing methods, such as the highly discriminatory pulsed-field gel electrophoresis (PFGE), has been attempted with various markers and typing schemes, including spa typing and multilocus sequence typing. However, despite a number of advantages, none of these methods showed convincing evidence for performance in epidemiological typing comparable to that of PFGE. By using three sets of 48 MRSA strains comprising isolates that were (i) genetically highly diverse, (ii) genetically related, and (iii) obtained from long-term carriers, we analyzed the performance of the four highly polymorphic S. aureus markers: clfA, clfB, fnbA, and spa. Typeability, discriminatory power, in vivo stability, and evolution of these markers were compared to those of PFGE. Clearly, none of the markers alone could match the discriminatory power of PFGE (63 genotypes; index of discrimination of 0.96). Instead, this could be achieved by combining markers in pairs. We showed that by using only 3' partial sequences of approximately 500 bp, the majority of each marker's discriminatory power was displayed, and using the partial sequences, the best performance was obtained with the combination of clfB and spa (57 genotypes; index of discrimination of 0.94). Genetic changes were not observed for any of the sequence markers over a period of 3 years and in the case of partial sequences for a period of more than 4 years. This is in contrast to PFGE where changes occurred after several months. The genetic differences found between isolate pairs of long-term carriers and among highly related isolates indicated clonal evolution. A typing scheme based on 500-bp 3' partial sequences of clfB and spa is proposed.

FIG. 1.

Schematic representation of clfA, clfB, fnbA, and spa, constructed on the basis of data from a previous report (8). Boxes indicate segments of each gene with signal sequences (S), wall-spanning regions (W), repeated and conserved wall-spanning regions (W_R and W_C, respectively), and membrane-spanning regions (M). Boxes labeled A to E are domains that were previously assigned. Repeated segments are indicated with vertical black and white shadings (each segment represents one repeat motif), whereas regions with small repeat motifs are indicated with horizontal black and white shading. Small arrows indicate primer annealing sites located in conserved regions flanking the repeat-containing regions. The different sequences of regions used for analyses are indicated by lines above the boxes. Continuous black lines represent the whole amplified repeat-containing regions (clfA, clfB, fnbA, and spa), long arrows indicate 500-bp partial sequences of the 3′ or 5′ region of the genes (3p500 or 5p500, respectively) which were taken for analysis. PCR amplification products varied in size mostly due to variation in repeat number; thus, it is possible that partial 5′ and 3′ sequences overlap.