Using Whole Genome Analysis to Examine Recombination across Diverse Sequence Types of Staphylococcus aureus

Abstract

Staphylococcus aureus is an important clinical pathogen worldwide and understanding this organism's phylogeny and, in particular, the role of recombination, is important both to understand the overall spread of virulent lineages and to characterize outbreaks. To further elucidate the phylogeny of S. aureus, 35 diverse strains were sequenced using whole genome sequencing. In addition, 29 publicly available whole genome sequences were included to create a single nucleotide polymorphism (SNP)-based phylogenetic tree encompassing 11 distinct lineages. All strains of a particular sequence type fell into the same clade with clear groupings of the major clonal complexes of CC8, CC5, CC30, CC45 and CC1. Using a novel analysis method, we plotted the homoplasy density and SNP density across the whole genome and found evidence of recombination throughout the entire chromosome, but when we examined individual clonal lineages we found very little recombination. However, when we analyzed three branches of multiple lineages, we saw intermediate and differing levels of recombination between them. These data demonstrate that in S. aureus, recombination occurs across major lineages that subsequently expand in a clonal manner. Estimated mutation rates for the CC8 and CC5 lineages were different from each other. While the CC8 lineage rate was similar to previous studies, the CC5 lineage was 100-fold greater. Fifty known virulence genes were screened in all genomes in silico to determine their distribution across major clades. Thirty-three genes were present variably across clades, most of which were not constrained by ancestry, indicating horizontal gene transfer or gene loss.

Fig 1. Evolutionary relationships of S. aureus clones found in the US and abroad.

(A) Circular map indicating homoplasy and SNP density in all taxa. Outer grey circle indicates the core reference chromosome beginning at the origin. The external track indicates the homoplasy density and the internal tack indicates parsimony informative (PI) SNPs for all strains analyzed showing dispersed homoplasy and PI SNP density. (B) Maximum-parsimony tree of 64 isolates of diverse S. aureus based on 80,836 SNPS, of which 57,236 were parsimony-informative, with a consistency index of 0.59. The numbers shown next to the branches represent the percentage of replicate trees where associated taxa cluster together based on 100 bootstrap replicates. The tree is rooted with the CC45 clade. Clonal complex or sequence types are indicated. Circular maps of homoplasy and SNP density for four of the clonal complexes and one sequence type (ST239) are located to the right of the tree and those for five of the branches are located on the left of the tree.

Fig 2

(A) Maximum-parsimony tree of the 16 strains belonging to the CC8 clade based on 1,717 SNPS (860 parsimony informative SNPs) with a consistency index of 0.99. (B) Maximum-parsimony tree of the 17 strains belonging to the CC5 clade based on 1,948 SNPS (632 parsimony informative SNPs) with a consistency index of 0.76.

Fig 3. Heat map indicating the BSR values for virulence genes that varied across strains (33 of 50 screened).

BSR values visualized with Multi-Experiment Viewer [27]. The left hand side of the figure contains the phylogeny as in Fig 1A. Clades are indicated with boxes; the black boxes indicated the CC5 and CC8 groups and the red boxes indicate the USA300 and USA100 groups. Gene names along with scale for heat map are indicated at the top of the heat map.