ALT (A) Images of the unstable colony phenotype of N315 L3 passage cycle 32, with hyper-pigmented SCV and large white colonies. (B) Mutations identified in N315 L3 isolates from passage cycle 4 (inner circle), 20 (middle circle), and 32 (outer circle). Mutations in corA and rsbW were identified and maintained since passage cycle 4. (C) Reversion of rsbW mutation to WT in N315 L3 isolate reverts the colony phenotype. D) Bacteria grown overnight at 37°C in nutrient-rich media prior to infection. L3 orange and L3 white isolates showed different survival in human blood, intracellular survival (after 72 h with gentamicin), and extracellular growth in THP-1 cultures (after 24 h without antibiotics) to parental strain and L3::rsbWWT. (E) L3 white achieves lower bacterial levels than parental or L3 orange in a zebrafish embryo infection model. (F) L3 orange isolate has an intermediate vancomycin resistance phenotype.