Ga-PpIX-Mediated Photodynamic Inactivation of <i>Staphylococcus aureus</i> Small-Colony Variants

Small-colony variants (SCVs) are subtypes found in persistent or chronic infections that are highly tolerant to oxidative or antibiotic stress but revert to a normal phenotype under standard conditions. Here, we show that SCVs of <i>Staphylococcus aureus</i> are susceptible to targeted antimicrobial photodynamic inactivation (aPDI) using a non-iron hemin analog (Ga-PpIX) as a photosensitizer. <i>S. aureus</i> (PCI 1203) cultivated with low levels of H₂O₂ yields reproducible SCVs in either standard (Fe-replete) or Fe-deficient media (R-SCV or D-SCV, respectively). R-SCVs are hemin auxotrophs whose acquisition systems operate independently of the ferric iron regulator (Fur), based on flow cytometry analysis. aPDI treatment of R-SCVs using a 405 nm light source produces a 3-log reduction in CFU/mL using as little as 1.5 μM Ga-PpIX, whereas <i>S. aureus</i> cultured under standard conditions is unreceptive. D-SCVs have much lower affinity for hemin yet are highly susceptible to aPDI at nanomolar levels of Ga-PpIX (3-log reduction at 0.06 μM). SCVs generated through oxacillin stress (O-SCVs) are also susceptible to aPDI (3-log reduction at 5 μM Ga-PpIX), attributable in part to heightened ROS sensitivity. We conclude that <i>S. aureus</i> SCVs can be more susceptible to Ga-PpIX-mediated aPDI than their wild-type counterparts, albeit for diverse reasons, providing a basis for developing light-mediated antimicrobial therapies to combat persistent <i>S. aureus</i> infections.