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 (^(1)O_2, produced via Type II mechanism) and hydroxyl radical (HO., produced via Type I mechanism). This study examined the relative contributions of ^(1)O_2 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 ^(1)O_2 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 (NaN_3) is a quencher of ^(1)O_2. 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 ^(1)O_2 (quenched by NaN_3), while BB6 produced HO. in addition to ^(1)O_2 when NaN_3 potentiated probe activation. NaN_3 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 NaN_3 dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN_3 almost failed to inhibit killing of Gram-positive bacteria. Conclusion: Azidyl radicals may be formed from NaN_3 and HO.. It may be that Gram-negative bacteria are more susceptible to HO. while Gram-positive bacteria are more susceptible to ^(1)O_2. The differences in NaN_3 inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN_3 into cell walls of bacteria.