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

## TL;DR

This study explores how different photosensitizers generate reactive oxygen species to kill MRSA bacteria using light therapy, identifying which types of damage are most effective.

## Contribution

The first characterization of ROS profiles and damage pathways for multiple photosensitizers in antimicrobial photodynamic therapy against MRSA.

## Key findings

- Seven photosensitizers generated distinct reactive oxygen species profiles in MRSA treatment.
- Singlet oxygen was primarily responsible for damage from PC and GM, while superoxide and hydroxyl radicals were key for GP, QC, and MB.
- Combining photosensitizers with different mechanisms enhanced MRSA inhibition at lower concentrations.

## Abstract

Methicillin-resistant Staphylococcus aureus (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 Passiflora cincinnata 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.