Comparative genomics of Staphylococcus aureus musculoskeletal isolates

Abstract

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.

FIG. 1.

Dendrogram illustrating genetic relationships between strains. The dendrogram was derived from PFGRC array data based on Spotfire hierarchical clustering. MLST for UAMS-1, UAMS-601, and RN6390 was performed according to the method of Maiden et al. (38). The spa genes for UAMS-1, UAMS-601, and RN6390 were amplified, sequenced, and typed according to the method of Shopsin et al. (62). Agr subgroups were determined by direct sequence analysis of the agr locus. The percentage of genes present or absent in each strain by comparison to UAMS-1 was determined from final PFGRC microarray results.

FIG. 2.

Southern hybridization for validation of array data. Genomic DNA isolated from the strain indicated above each lane was hybridized with probes corresponding to the genes shown to the right of each panel. The sarU probe used in these experiments also included a region corresponding to sarT, while the sarT probe was limited to sarT itself.

FIG. 3.

PCR verification of fnbA and fnbB. Genomic DNA isolated from the indicated strains was used as a template in PCRs with primers that amplify unique regions of fnbA or fnbB. The results observed with UAMS-1, COL, and N315 are representative of the results observed with the other seven strains as described in the text.

FIG. 4.

Microtiter plate assay of biofilm formation. Biofilm formation was assessed as described by Beenken et al. (5). Results are shown as absorbance at 560 nm (A₅₆₀) and represent the average and standard errors of the means from nine independent experiments. Strain U929 (UAMS-1 sarA mutant) was included as a negative control. Distribution of cna and sasG among each of the 10 strains was confirmed by microarray results, Southern hybridization, and BLAST searches. Capsular serotype was determined from microarray results for all strains, and confirmed in UAMS-1 by serotyping analysis (Chia Y. Lee, personal communication). The designation “d” indicates that MW2 and SANGER-476 contain a divergent form of sasG (53).