Improved Subtyping of Staphylococcus aureus Clonal Complex 8 Strains Based on Whole-Genome Phylogenetic Analysis

Abstract

Strains of Staphylococcus aureus in clonal complex 8 (CC8), including USA300, USA500, and the Iberian clone, are prevalent pathogens in the United States, both inside and outside health care settings. Methods for typing CC8 strains are becoming obsolete as the strains evolve and diversify, and whole-genome sequencing has shown that some strain types fall into multiple sublineages within CC8. In this study, we attempt to clarify the strain nomenclature of CC8, classifying the major strain types based on whole-genome sequence phylogenetics using both methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) genomes. We show that isolates of the Archaic and Iberian clones from decades ago make up the most basal clade of the main CC8 lineages and that at least one successful lineage of CC8, made up mostly of MSSA, diverged before the other well-known strain types USA500 and USA300. We also show that the USA500 type includes two clades separated by the previously described "Canadian epidemic MRSA" strain CMRSA9, that one clade containing USA500 also contains the USA300 clade, and that the USA300-0114 strain type is not a monophyletic group. Additionally, we present a rapid, simple CC8 strain-typing scheme using real-time PCR assays that target single nucleotide polymorphisms (SNPs) derived from our CC8 phylogeny and show the significant benefit of using more stable genomic markers based on evolutionary lineages over traditional S. aureus typing techniques. This more accurate and accessible S. aureus typing system may improve surveillance and better inform the epidemiology of this very important pathogen.IMPORTANCEStaphylococcus aureus is a major human pathogen worldwide in both community and health care settings. Surveillance for S. aureus strains is important to our understanding of their spread and to informing infection prevention and control. Confusion surrounding the strain nomenclature of one of the most prevalent lineages of S. aureus, clonal complex 8 (CC8), and the imprecision of current tools for typing S. aureus make surveillance and source tracing difficult and sometimes misleading. In this study, we clarify the CC8 strain designations and propose a new typing scheme for CC8 isolates that is rapid and easy to use. This typing scheme is based on relatively stable genomic markers, and we demonstrate its superiority over traditional typing techniques. This scheme has the potential to greatly improve epidemiological investigations of S. aureus.

Keywords: CC8; MRSA; MSSA; Staphylococcus aureus; assay development; phylogeny; strain typing; whole genome.

FIG 1

WGS-based maximum likelihood phylogeny (using the best-fit model TVMe+ASC) of 497 S. aureus isolate genomes showing the CC8 group in the context of the whole of S. aureus. This analysis includes 1,000 bootstraps of 275,242 total SNPs in a core genome size (the length of the reference genome covered by all samples, excluding repeated regions) of 1.84 Mbp. Regions of chromosomal exchange among lineages resulting in hybrid strains (e.g., ST239) were not excluded. Bootstrap values are 100%, except where indicated. Branches of the phylogeny on which SNPs were selected for assay development are marked with a red triangle.

FIG 2

WGS-based maximum likelihood phylogeny (using the best-fit model TVMe+ASC) of 348 genomes of S. aureus (229 MRSA and 119 MSSA) belonging to the Inner CC8 clade (excluding ST239 and ST630 genomes), illustrating the relationship structure of clinically important CC8 groups and showing that genetic marker inference (GMI) strain typing is not always indicative of genetic relationship. MSSA genomes, on branches colored orange (Hex DF5000, RGB [223,80,0]), are interspersed among MRSA genomes. This analysis includes 1,000 bootstraps of 13,988 SNPs. Nodes with bootstrap values of <90% are marked with purple triangles. The core genome size is 2.26 Mbp (78.8% of reference genome FPR3757). Branches of the phylogeny on which SNPs were selected for assay development are marked with a red triangle.

FIG 3

The genetic marker inference (GMI) methodology used for inferring S. aureus strain types by using genetic markers.