Characterization of the Oxidative Profile, Damage Pathways, and Synergism of Photosensitizers in Antimicrobial Photodynamic Therapy against Methicillin-Resistant <i>Staphylococcus aureus</i>

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is an opportunistic pathogen that is difficult to treat due to its resistance to multiple classes of antibiotics. An alternative control method is antimicrobial photodynamic therapy (aPDT), which combines photosensitizing compounds (PS) and light to generate reactive oxygen species (ROS). Despite its effectiveness, the damaging pathways associated with ROS have been poorly explored. To address this gap, the present study investigated the ROS generation profile (O₂•^-, •OH, and ¹O₂), lipid peroxidation levels, and the role of these reactive species in the inhibition of MRSA in aPDT mediated by seven PSs: curcumin (CM), green propolis (GP), butanolic fraction of <i>Passiflora cincinnata</i> extract (PC), quercetin (QC), malachite green (GM), methylene blue (MB), and toluidine blue (TBO). The effect of combining PS with different photochemical mechanisms was also investigated. Photodynamic antimicrobial activity was observed for all PS, which reduced or inhibited MRSA growth. Under light irradiation, PS presented different ROS production profiles: CM (¹O₂), GP (•OH), PC (•OH and ¹O₂), QC (O₂•^-), GM (O₂•^-, ¹O₂), MB (•OH, ¹O₂), and TBO (¹O₂). However, not all generated ROS contributed to bacterial inhibition. It was determined that ¹O₂ is the primary species responsible for the oxidatively generated damage induced by PC and GM against MRSA, whereas O₂•^- and •OH are essential for the inhibitory activity of GP, QC, and MB. Combining these data with lipid peroxidation levels in aPDT indicates that the damage caused by CM, GM, MB, and TBO is associated with the oxidation of unsaturated lipids, probably through the formation of primary lipid peroxidation products such as lipid radicals and hydroperoxides. The lack of increased lipid peroxidation in MRSA in aPDT mediated by GP, QC, and PC indicates that oxidative damage is directed toward other biomolecules. We demonstrated that different photochemical mechanisms can complement each other, enabling a reduction in the concentrations initially tested for one of the PS combined treatments. The combinations PC-CM, PC-GP, and GM-TBO were able to inhibit MRSA growth in aPDT. This study is the first to characterize the ROS profile of multiple PS and associate specific ROS damage pathways in aPDT against MRSA.