Antimicrobial blue light inactivation of Candida albicans: In vitro and in vivo studies

Abstract

Fungal infections are a common cause of morbidity, mortality and cost in critical care populations. The increasing emergence of antimicrobial resistance necessitates the development of new therapeutic approaches for fungal infections. In the present study, we investigated the effectiveness of an innovative approach, antimicrobial blue light (aBL), for inactivation of Candida albicans in vitro and in infected mouse burns. A bioluminescent strain of C. albicans was used. The susceptibilities to aBL (415 nm) were compared between C. albicans and human keratinocytes. The potential development of aBL resistance by C. albicans was investigated via 10 serial passages of C. albicans on aBL exposure. For the animal study, a mouse model of thermal burn infected with the bioluminescent C. albicans strain was used. aBL was delivered to mouse burns approximately 12 h after fungal inoculation. Bioluminescence imaging was performed to monitor in real time the extent of infection in mice. The results obtained from the studies demonstrated that C. albicans was approximately 42-fold more susceptible to aBL than human keratinocytes. Serial passaging of C. albicans on aBL exposure implied a tendency of reduced aBL susceptibility of C. albicans with increasing numbers of passages; however, no statistically significant difference was observed in the post-aBL survival rate of C. albicans between the first and the last passage (P>0.05). A single exposure of 432 J/cm(2) aBL reduced the fungal burden in infected mouse burns by 1.75-log10 (P=0.015). Taken together, our findings suggest aBL is a potential therapeutic for C. albicans infections.

Keywords: antimicrobial blue light; bioluminescence imagining; burn; candida albicans; endogenous photosensitizer; mouse model.

Figure 1.

Antimicrobial blue light inactivation of C. albicans and human keratinocytes in vitro. Bars: standard deviation. The mean inactivation rate coefficients (k_H) of C. albicans and keratinocytes were 0.0795 and 0.0019 cm²/J, respectively, indicating an approximately 42-fold faster inactivation rate of C. albicans by aBL than keratinocytes.

Figure 2.

TEM images of C. albicans cells. (A) Untreated C. albicans cells: N=nucleus; V = vacuole; M=mitochondria; CM = cytoplasmic membrane; CW = cell wall. (B)-(D) aBL-treated C. albicans cells: (B) Decomposition of inner organelles with deformed cell wall (aBL radiant exposure = 35.1 J/cm²); (C) Unusual vacuole growth, driving electron-dense structures to the cell periphery (aBL radiant exposure = 35.1 J/cm²); and (D) Complete loss of cytoplasmic components with disrupted cell wall (aBL radiant exposure = 70.2 J/cm²).

Figure 3.

Fluorescence emission spectra of lysed C. albicans cells in NaOH/SDS: (A) Excitation at 405 nm led to porphyrin-like dual fluorescence emission maxima at 631 and 667 nm; (B) Excitation at 470 nm gave rise to a flavin-like emission maximum at 515 nm.

Figure 4.

Change of extent of aBL inactivation of C. albicans (log₁₀ CFU) with numbers of passages of C. albicans on aBL exposure. Bars: standard deviation. No statistically significant difference was observed in aBL inactivation extent between the 1st and 10th passage (P = 0.09).

Figure 5.

(A-B) Successive fungal luminescence images of representative mouse burns infected with 10⁵ CFU of bioluminescent C. albicans, with (panel A) and without aBL therapy (panel B), respectively. aBL irradiance = 90 mW/cm². aBL was delivered at 12 h after fungal inoculation. In panel A, the Day 1-0 J/cm² image was taken just prior to aBL exposure; Day 1-27 J/cm², Day 1- 54 J/cm², Day 1-81 J/cm², Day 1-108 J/cm², Day 1-162 J/cm², Day 1-216 J/cm², Day 1-324 J/cm² and Day 1- 432 J/cm² images were taken immediately after respective aBL exposure had been delivered; and the Day 1 to Day 8 images were taken at Day 1 to Day 8 after fungal inoculation, respectively. In panel B, the Day 1 to Day 8 images were taken at Day 1 to Day 8 after fungal inoculation, respectively. (C) Dose responses of mean fungal luminescence of mouse burns infected with 10⁵ CFU of C. albicans, with (n = 8) and without (n = 8) aBL therapy, respectively. aBL was delivered at 12 h after fungal inoculation. Bars: standard deviation.

Figure 6.

(A) Time courses of mean fungal luminescence (RLU, day 2 to day 11) of mouse burns infected with 10⁵ CFU of C. albicans, with (n=8) and without (n=8) aBL therapy, respectively. aBL was delivered at 12 h after fungal inoculation. Bars: standard deviation. (B) Mean areas under the fungal luminescence versus time curves in the two-dimensional coordinate system in panel A, representing the overall fungal burden of infected mouse burns. Bars: standard deviation.