title: Type I and Type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Infectious Diseases, First Affiliated College & Hospital, Guangxi Medical University, Nanning 530021, China.
authors: Liyi
authors: L
authors: Huang
authors: Yi
authors: Y
authors: Xuan
authors: Yuichiro
authors: Y
authors: Koide
authors: Timur
authors: T
authors: Zhiyentayev
authors: Masamitsu
authors: M
authors: Tanaka
authors: Michael R
authors: MR
authors: Hamblin
mesh_terms: D005456:Fluorescent Dyes; D037741:Fullerenes / Q000494:pharmacology; D006090:Gram-Negative Bacteria / Q000187:drug effects* / Q000378:metabolism; D006094:Gram-Positive Bacteria / Q000187:drug effects* / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D066298:In Vitro Techniques; D050296:Microbial Viability / Q000187:drug effects*; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011094:Polyethyleneimine / Q000031:analogs & derivatives / Q000494:pharmacology; D011108:Polymers / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D026082:Singlet Oxygen / Q000378:metabolism; D019810:Sodium Azide / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D037741:Fullerenes; D017319:Photosensitizing Agents; D011108:Polymers; D017382:Reactive Oxygen Species; D026082:Singlet Oxygen; D017665:Hydroxyl Radical; D011094:Polyethyleneimine; D019810:Sodium Azide