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. Lasers Surg. Med. 44: 490–499, 2012. © Wiley Periodicals, Inc.