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.

Figure 1. Chemical structures and absorption spectra

(A) PEI-ce6; (B) BB6; (C) Absorption spectra recorded at 10 µM in H₂O or DMA together with emission spectra of the 400—700-nm white light and the 660+/−15-nm red light source.

Figure 2. Fluorescence ROS probe studies with photoactivated PEI-ce6 and BB6

Fluorescence generated from probes (10 µM) and PS (2 µM) in water without addition, and with addition of 10 mM NaN3 or 100 mM DMT. (A) BB6 and SOSG, white light; (B) BB6 and HPF, white light; (C) PEI-ce6 and SOSG, 660-nm light; (D) PEI-ce6 and HPF, 660-nm light. The fluences of white light have been corrected to show photons absorbed by the BB6.

Figure 3. HPF fluorescence probe study with Fenton reagent

Stated concentrations of equimolar FeSO₄ and H₂O₂ were added to 10 µM HPF without addition or with addition of 10 mM NaN3 or 200 mM DMT and fluorescence read immediately.

Figure 4. Killing of bacteria by Fenton reagent and effect of NaN₃

Suspensions of bacteria (10(8)/mL) had stated concentrations of equimolar FeSO₄ and H₂O₂ added, with or without the addition of 0.5 mM NaN₃ and after 60 min aliquots were removed for CFU determination. *** P<0.001; ** P<0.01 one way ANOVA. (A) S. aureus; (B) E. faecalis; (C) E. coli; (D) P. aeruginosa; (E) P. mirabilis.

Figure 5. APDT of bacteria mediated by PEI-ce6 and effect of NaN₃

Suspensions of bacteria (10(8)/mL) were incubated with PEI-ce6 for 30 min followed by addition of NaN₃ (0–10 mM) and illumination with 660-nm light and CFU determination. (A) 400-nM PEI-ce6 and S. aureus; (B) 400-nM PEI-ce6 and E. faecalis; (C) 10 µM PEI-ce6 and E. coli; (D) 10 µM PEI-ce6 and P. aeruginosa; (E) 10 µM PEI-ce6 and P. mirabilis.

Figure 6. APDT of bacteria mediated by BB6 and effect of NaN₃

Suspensions of bacteria (10(8)/mL) were incubated with BB6 for 30 min followed by addition of NaN₃ (0–10 µM) and illumination with white light (400–700-nm) and CFU determination. (A) 1 µM BB6 and S. aureus; (B) 1 µM BB6 and E. faecalis; (C) 2 µM BB6 and E. coli; (D) 4 µM BB6 and P. aeruginosa; (E) 4 µM BB6 and P. mirabilis.