Colonizing and infecting subclones diverge during Staphylococcus aureus infection

Therapy of Staphylococcus aureus bacteremia is often ineffective, even under optimal conditions, and adapted subclones with attenuated agr-mediated virulence activation are associated with persistent infection and mortality. To understand how apparent loss of virulence leads to increased mortality, we sequenced complete genomes from clone pairs from colonizing and infected sites of patients in whom S. aureus demonstrated a within-host downshift in agr function in the infecting isolate. Clone pairs with a downshift in agr function showed substantial genetic divergence compared to wild-type pairs from controls. Complementation studies further identified an agr-defective bacteremic strain that was highly virulent in vivo, which we linked to a mutation that restored expression of the agr-regulated ess/Type-VII secretion system; a known virulence factor. Our results suggest that selection pressure during invasive infection is strong enough to mutationally bypass agr-deficiency associated loss of virulence, and that efforts to suppress agr function may need to be reconsidered. Clonal S. aureus isolates from colonizing (nares) and infecting sites (blood) were obtained from a single patient. Whole-genome comparisons of single colonies from both isolates revealed 361 genetic variants between strain genomes, including a loss of agr functionality in the blood strain. To assess the impact of non-agr mutations engineered an agr knockout of the wild-type nares strain and complemented the naturally occurring agr mutant blood strain. Wild-type agr genes were transduced in single copy to the naturally occurring agr-defective variant using the staphylococcal pathogenicity island (SaPI)-1 attC locus as the insertion site. The four different strains were then grown to late log (OD600 of ~0.8) and profiled by directional RNA-Seq, in duplicate.