Unstable chromosome rearrangements in <i>Staphylococcus aureus</i> cause phenotype switching associated with persistent infections

Abstract

<i>Staphylococcus aureus</i> small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable <i>S. aureus</i> SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of <i>S. aureus</i> linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the <i>S. aureus</i> chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete <i>S. aureus</i> genomes showed strong signatures of recombination between <i>hsdMS</i> genes, suggesting that analogous CI has repeatedly occurred during <i>S. aureus</i> evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major <i>S. aureus</i> lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in <i>S. aureus</i> associated with SCV generation and persistent infections.