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

Abstract 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. NaN 3 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. Lasers Surg. Med. 44: 490–499, 2012. © Wiley Periodicals, Inc.