Lethal photosensitization of wound-associated microbes using indocyanine green and near-infrared light

Abstract

Background: The increase in resistance to antibiotics among disease-causing bacteria necessitates the development of alternative antimicrobial approaches such as the use of light-activated antimicrobial agents (LAAAs). Light of an appropriate wavelength activates the LAAA to produce cytotoxic species which can then cause bacterial cell death via loss of membrane integrity, lipid peroxidation, the inactivation of essential enzymes, and/or exertion of mutagenic effects due to DNA modification. In this study, the effect of the LAAA indocyanine green excited with high or low intensity light (808 nm) from a near-infrared laser (NIR) on the viability of Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa was investigated.

Results: All species were susceptible to killing by the LAAA, the bactericidal effect being dependent on both the concentration of indocyanine green and the light dose. Indocyanine green photosensitization using both high (1.37 W cm(-2)) and low (0.048 W cm(-2)) intensity NIR laser light was able to achieve reductions of 5.6 log10 (>99.99%) and 6.8 log10 (>99.99%) in the viable counts of Staph. aureus and Strep. pyogenes (using starting concentrations of 106-107 CFU ml(-1)). Kills of 99.99% were obtained for P. aeruginosa (initial concentration 108-109 CFU ml(-1)) photosensitized by the high intensity light (1.37 W cm(-2)); while a kill of 80% was achieved using low intensity irradiation (0.07 W cm(-2)). The effects of L-tryptophan (a singlet oxygen scavenger) and deuterium oxide (as an enhancer of the life span of singlet oxygen) on the survival of Staph. aureus was also studied. L-tryptophan reduced the proportion of Staph. aureus killed; whereas deuterium oxide increased the proportion killed suggesting that singlet oxygen was involved in the killing of the bacteria.

Conclusion: These findings imply that indocyanine green in combination with light from a near-infrared laser may be an effective means of eradicating bacteria from wounds and burns.

Figures

Figure 1

High intensity lethal photosensitization of Staph. aureus □ and Strep. pyogenes ▨ with 25 μg ml-1 ICG or P. aeruginosa ■ with 200 μg ml-1 ICG. An equal volume of either 50 μg ml-1 (in the case of Staph. aureus and Strep. pyogenes) or 400 μg ml-1 (in the case of P. aeruginosa) ICG (L-S+ and L+S+) or PBS (L-S- and L+S-) was added to each bacterial suspension, samples were left for 10 minutes in the dark and then irradiated at a fluence rate of 1.37 W cm-2 with a light dose of 411 J cm-2 from a NIR 808 nm laser (L+S- and L+S+) or kept in the dark (L-S+ and L-S-).

Figure 2

Lethal photosensitization of Staph. aureus ●, Strep. pyogenes □ with 25,50, 100 and 200 μg ml-1 ICG, and P. aeruginosa ▲ with 25,50, 100, 200 and 250 μg ml-1 ICG. An equal volume of the appropriate ICG concentration (L+S+) was added to each bacterial suspension, samples were left for 10 minutes in the dark and then irradiated at a fluence rate of 1.37 W cm-2 with light dose of 411 J cm-2 from a NIR 808 nm laser.

Figure 3

(A) Lethal photosensitization of Staph. aureus □ and Strep. pyogenes using a final concentration of 25 μg ml-1 ICG. Samples were left for 10 minutes in the dark and then irradiated using a fluence rate of 0.048 W cm-2 for 15 and 30 minutes, corresponding to light doses of 43 & 86 J cm-2 respectively. (B) Lethal photosensitization of P. aeruginosa ■ using a final concentration of 200 μg ml-1 ICG. Samples were left for 10 minutes in the dark and then irradiated using a fluence rate of 0.07 W cm-2 for 35 minutes, corresponding to light dose of 147 J cm-2. L-S- and L+S- were kept in the dark.

Figure 4

High intensity lethal photosensitization of Staph. aureus □, Strep. pyogenes ▨ with 25 μg ml-1 ICG and P. aeruginosa ■ with 200 μg ml-1 ICG. An equal volume of either 50 μg ml-1 (in the case of Staph. aureus and Strep. pyogenes) or 400 μg ml-1 (in the case of P. aeruginosa) ICG (L-S+ and L+S+) or PBS (L-S- and L+S-) was added to each bacterial suspension. Samples were left in the dark for 30 minutes and then (L+S- and L+S+) irradiated at a fluence rate of 1.37 W cm-2 for 1, 3 or 5 minutes with light from a NIR 808 nm laser, corresponding to light doses of 82, 247, or 411 J cm-2 respectively. Samples L-S+ and L-S- were kept in the dark.

Figure 5

Lethal photosensitization of Staph. aureus in 50% horse serum (HS) using 25 □ or 200 ■ μg ml-1 ICG. An equal volume of either 50 or 400 μg ml-1 ICG (L-S+ and L+S+) or PBS (L-S- and L+S-) was added to Staph. aureus suspensions in HS and samples were left for 10 minutes in the dark and then irradiated at a fluence rate of 1.37 W cm-2 and a light dose of 411 J cm-2 using a NIR 808 nm laser (L+S- and L+S+) or kept in the dark (L-S+ and L-S-).

Figure 6

High intensity lethal photosensitization of Staph. aureus (A) suspended in 10 mM L-tryptophan ■ or suspended in H2O and (B), suspended in D2O or suspended in H2O □. In this experiment an ICG concentration of 25 μg ml-1 was used and samples were pre-incubated in the dark for 10 minutes before being irradiated at a fluence rate of 1.37 W cm-2 and light dose of 82 J cm-2.