A Novel Method to Characterize in vitro Interactions between Photosensitizers and Antimicrobials using a Modified Checkerboard Assay

Antimicrobial photodynamic therapy (aPDT) combines a photosensitizer with light and molecular oxygen to generate reactive oxygen species that damage microbial cells. Using complimentary mechanisms of action, the sublethal cellular damages caused by aPDT have shown priming effects that enhance microbial sensitivity to standard antimicrobial chemotherapy, offering a potent and resistance-free approach. To determine the types of interaction between aPDT and antimicrobial compounds—whether synergistic, additive, indifferent, or antagonistic—we developed a modified checkerboard assay based on ASM and CLSI guidelines. First, a two-dimensional concentration gradient of the PS and the antimicrobial agent is prepared and irradiated in 96-well plates. Following an incubation period, optical density data obtained by a plate reader is used in a custom-made calculator that automatically determines the minimum inhibitory concentrations and fractional inhibitory concentration indexes. This approach provides basis for a standard method that objectively characterizes the type of interaction between aPDT and antimicrobial compounds.