Genome-wide analysis of ruminant Staphylococcus aureus reveals diversification of the core genome

Abstract

Staphylococcus aureus causes disease in humans and a wide array of animals. Of note, S. aureus mastitis of ruminants, including cows, sheep, and goats, results in major economic losses worldwide. Extensive variation in genome content exists among S. aureus pathogenic clones. However, the genomic variation among S. aureus strains infecting different animal species has not been well examined. To investigate variation in the genome content of human and ruminant S. aureus, we carried out whole-genome PCR scanning (WGPS), comparative genomic hybridizations (CGH), and the directed DNA sequence analysis of strains of human, bovine, ovine, and caprine origin. Extensive variation in genome content was discovered, including host- and ruminant-specific genetic loci. Ovine and caprine strains were genetically allied, whereas bovine strains were heterogeneous in gene content. As expected, mobile genetic elements such as pathogenicity islands and bacteriophages contributed to the variation in genome content between strains. However, differences specific for ruminant strains were restricted to regions of the conserved core genome, which contained allelic variation in genes encoding proteins of known and unknown function. Many of these proteins are predicted to be exported and could play a role in host-pathogen interactions. The genomic regions of difference identified by the whole-genome approaches adopted in the current study represent excellent targets for studies of the molecular basis of S. aureus host adaptation.

FIG. 1.

MLST and PFGE genotyping of the 12 S. aureus strains. (A) Radial tree depicting the phylogenetic relationships of the STs of the 12 strains used in this study and all of the S. aureus STs available in the MLST database (1,095 as of March 2008). MEGA 4.0 software and the neighbor-joining method with a bootstrap value of 1,000 were used to build the tree. (B) PFGE macrorestriction analysis of the chromosome of the 12 strains (upper). Dendrogram presenting the percentage of genetic similarity between the 12 strains (lower). The unweighted-pair group method using average linkages and a Dice coefficient (with a tolerance limit of 1.1%) were used to build the dendrogram. M, molecular size marker.

FIG. 2.

Summary and data comparison of the WGPS and the microarray analyses of the set of 12 S. aureus strains. Results of the WGPS of the 12 strains are shown in the upper lines. WGPS scanning analysis is presented as follows. Segments that yielded PCR products of identical, smaller, and bigger sizes compared to those in N315 are depicted by blue, dark blue, and light blue rectangles, respectively. Fragments that were not amplified are depicted by white rectangles. Fragments that were demonstrated as being absent by framing PCR experiments are depicted in gray. The positions of the MGEs, as determined in the N315 annotation, are given in the row above WGPS results. Results from the gene content analysis using the whole-genome microarray are shown in the lower lines. CGH analyses were carried out using N315 as the reference (Mu50, whose gene content is known, was not analyzed here). Genes that gave a signal in both N315 and the test strain were considered present and are indicated in blue. Genes that gave a low signal or no signal at all compared to that of N315 were judged as variable and are indicated in orange and red, respectively. The lower part of the figure summarizes the success rates of the WGPS. For each segment, the number of strains (1 to 12) that yielded an LR-PCR product is given.

FIG. 3.

Demonstration of host specificity for six ovine-caprine traits. PCR tests were run on 40 different strains isolated from human (hum) (8), bovine (bov) (16), and ovine-caprine (ov-cap) (16) hosts. PCR products were analyzed by electrophoresis on agarose gels. Host origins are indicated at the top of each gel. In the gels on the right, M indicates the RaoulI linear DNA marker (Appligene; Qbiogene, Strasbourg, France). In the gels on the left, M indicates a 2-log ladder linear DNA marker (New England Biolabs, Ozyme, France).