Type i and Type II mechanisms of antimicrobial photodynamic therapy: An in vitro study on gram-negative and gram-positive bacteria

Background and Objectives Antimicrobial photodynamic therapy (APDT) employs a non-toxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen ( ¹O₂, produced via Type II mechanism) and hydroxyl radical (HO^., produced via Type I mechanism). This study examined the relative contributions of ¹O₂ and HO^. to APDT killing of Gram-positive and Gram-negative bacteria. Study Design/Materials and Methods Fluorescence probes, 3'-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO^. and ¹O₂ produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI-ce6). Dimethylthiourea is a HO^. scavenger, while sodium azide (NaN₃) is a quencher of ¹O₂. Both APDT and killing by Fenton reaction (chemical generation of HO^.) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Results Conjugate PEI-ce6 mainly produced ¹O₂ (quenched by NaN₃), while BB6 produced HO^. in addition to ¹O₂ when NaN₃ potentiated probe activation. NaN₃ also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN₃ potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN₃ dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN₃ almost failed to inhibit killing of Gram-positive bacteria. Conclusion Azidyl radicals may be formed from NaN₃ and HO^.. It may be that Gram-negative bacteria are more susceptible to HO^. while Gram-positive bacteria are more susceptible to ¹O₂. The differences in NaN₃ inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN₃ into cell walls of bacteria.