Blue light rescues mice from potentially fatal Pseudomonas aeruginosa burn infection: efficacy, safety, and mechanism of action

Abstract

Blue light has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. However, the use of blue light for wound infections has not been established yet. In this study, we demonstrated the efficacy of blue light at 415 nm for the treatment of acute, potentially lethal Pseudomonas aeruginosa burn infections in mice. Our in vitro studies demonstrated that the inactivation rate of P. aeruginosa cells by blue light was approximately 35-fold higher than that of keratinocytes (P = 0.0014). Transmission electron microscopy revealed blue light-mediated intracellular damage to P. aeruginosa cells. Fluorescence spectroscopy suggested that coproporphyrin III and/or uroporphyrin III are possibly the intracellular photosensitive chromophores associated with the blue light inactivation of P. aeruginosa. In vivo studies using an in vivo bioluminescence imaging technique and an area-under-the-bioluminescence-time-curve (AUBC) analysis showed that a single exposure of blue light at 55.8 J/cm(2), applied 30 min after bacterial inoculation to the infected mouse burns, reduced the AUBC by approximately 100-fold in comparison with untreated and infected mouse burns (P < 0.0001). Histological analyses and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays indicated no significant damage in the mouse skin exposed to blue light at the effective antimicrobial dose. Survival analyses revealed that blue light increased the survival rate of the infected mice from 18.2% to 100% (P < 0.0001). In conclusion, blue light therapy might offer an effective and safe alternative to conventional antimicrobial therapy for P. aeruginosa burn infections.

Figures

Fig 1

Emission spectrum of Omnilux blue LED.

Fig 2

Dose response of blue light inactivation of P. aeruginosa (●) and keratinocytes (○) in vitro.

Fig 3

Transmission electron microscopy of P. aeruginosa cells. (A) Untreated P. aeruginosa cells. Bar = 100 nm. (B to D) P. aeruginosa cells after being exposed to 109.9 J/cm2 blue light: development of vacuoles within the cytoplasm (B) (bar = 100 nm), release of cytoplasmic material to the surrounding environment (C) (bar = 500 nm), and complete disappearance of cytoplasm (D) (bar = 100 nm).

Fig 4

Fluorescence spectra of P. aeruginosa cell pellets from overnight culture dissolved in NaOH-SDS. Excitation wavelength, 405 nm.

Fig 5

(A and B) Successive bacterial luminescence images of representative mouse burns infected with 3 × 106 CFU of luminescent P. aeruginosa with and without blue light (415 nm) exposure, respectively. Blue light was delivered at 30 min after bacterial inoculation. (C) Dose response of mean bacterial luminescence of mouse burns infected with 3 × 106 CFU of P. aeruginosa and exposed to blue light (415 nm) at 30 min after bacterial inoculation (n = 11). Bars, standard deviations. (D) Time courses of mean bacterial luminescence of the infected skin abrasions with (n = 11) and without (n = 11 at days 1 and 2, n = 4 at day 3) blue light exposure, respectively. Bars, standard deviations. RLU values in the blue light group versus RLU values in the untreated groups: day 1, P = 0.0008; day 2, P = 6.11 × 10−5; day 3, P = 0.049. (E) Mean areas under the bioluminescence versus time curves (from day 1 to day 2 in the two-dimensional coordinate system in panel D), representing the overall bacterial burden of mouse wounds. Bars, standard deviations. (F) Kaplan-Meier survival curves of blue light-treated (n = 11) and untreated (n = 11) mouse burns (P < 0.0001).

Fig 6

(A) Hematoxylin-and-eosin-stained histological sections of skin samples from a representative mouse exposed to blue light at a dose of 55.8 J/cm2. Skin samples were taken before blue light exposure and 0 h, 24 h, and 48 h after blue light exposure. Bar, 200 μm. (B) TUNEL analyses of DNA damage in the same mouse skin shown in panel A (100×). Skin samples were taken before blue light exposure and 0 h, 24 h, and 48 h after blue light exposure. Fluorescein and DAPI are represented by green and blue, respectively. DAPI was used for nuclear counterstaining. Arrows, TUNEL-positive cells.