New Insights into the Bacterial Targets of Antimicrobial Blue Light

Abstract

Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (λ = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD_99.9 = 54.7 J/cm²) relative to S. aureus (LD_99.9 = 158.9 J/cm²) and E. coli (LD_99.9 = 195 J/cm²). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (<LD₉₀) could damage the cell membrane in Gram-negative species but not in S. aureus. In all bacteria, oxidative damage to bacterial DNA (except P. aeruginosa), proteins, and lipids occurred after high aBL exposures (>LD_99.9). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms. IMPORTANCE Antimicrobial-drug development is facing increased scrutiny following the worldwide antibiotic crisis. Scientists across the world have recognized the urgent need for new antimicrobial therapies. In this sense, antimicrobial blue light (aBL) is a promising option due to its antimicrobial properties. Although aBL can damage different cell structures, the targets responsible for bacterial inactivation have still not been completely established and require further exploration. In our study, we conducted a thorough investigation to identify the possible aBL targets and gain insights into the bactericidal effects of aBL on three relevant pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This research not only adds new content to blue light studies but opens new perspectives to antimicrobial applications.

Keywords: endogenous chromophores; lipid peroxidation; membrane permeabilization; protein carbonylation; reactive oxygen species.

FIG 1

Dose-response curves for E. coli, P. aeruginosa and S. aureus exposed to aBL (λ = 410 ± 10 nm). Values are presented as means ± SEM.

FIG 2

Representative transmission electron micrographs illustrating aBL-induced ultrastructural damages in P. aeruginosa, S. aureus, and E. coli for LD₉₀ and LD_99.9. Red asterisk, agglutination of intracellular contents; black asterisk, cell wall/membrane damage; white arrow, leakage of intracellular contents; red arrow, membrane destabilization; red circle, elongation and morphological irregularities. Bars: 500 nm.

FIG 3

Porphyrin concentrations present within (a) P. aeruginosa, (b) S. aureus, and (c) E. coli. Symbols (•) represent the mean of three technical replicates. Data are presented as scatterplots with bar ± SEM.

FIG 4

FLIM images of endogenous fluorophores for P. aeruginosa (a to d), S. aureus (e to h), and E. coli (i to l). Different colors represent different fluorescence lifetimes for each bacterial species (green < blue < red). Homogeneous distribution of fluorophores can be observed for each lifetime decay. Shorter lifetimes are observed for E. coli. No fluorophores were detected in the septum of S. aureus for the shortest lifetime (e). Merged fluorescence signal (RGB) in each bacterial species (d, h, l) is also depicted.

FIG 5

Relative fluorescence units proportional to intracellular ROS production in P. aeruginosa (a), S. aureus (b), and E. coli (c) after aBL exposure. Symbols (•) represent the average of cumulative measurements over 1 h. Data are presented as scatterplots with bar ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to unirradiated control.

FIG 6

Effects of ROS scavengers on aBL killing of bacteria. Sodium azide (10 mM) and thiourea (150 μM) were used as scavengers of singlet oxygen and hydroxyl radicals, respectively. (a) P. aeruginosa, (b) S. aureus, and (c) E. coli. Symbols (•) represent the mean of three technical replicates. Data are presented as scatterplots with bar ± SEM. Different letters indicate significant differences between treatments (P < 0.05).

FIG 7

Analysis of DNA damage assayed by pulsed-field gel electrophoresis (PFGE) using the whole genome of P. aeruginosa (a), S. aureus (b), and E. coli (c). Bacterial samples were exposed to increasing radiant exposure of aBL (i.e., 0, 11.45, 22.9, 45.8, 91.6, 183.2, 366.4, and 549.6 J/cm²), followed by DNA extraction. For DNA cleavage, specific restriction endonucleases were used for each microorganism: Xbal, BcuI, and SmaI for E. coli, P. aeruginosa, and S. aureus, respectively. The reference molecular weight ladders for pulsed-field gel electrophoresis are identified by (L).

FIG 8

Effects of aBL on proteins in P. aeruginosa, S. aureus, and E. coli assessed by carbonyl protein quantification (a, b, and c) and total protein amount (d, e, and f). Symbols (•) represent the mean of three technical replicates. Data are presented as scatterplots with bar ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to unirradiated control.

FIG 9

Effects of aBL on lipids in (a) P. aeruginosa, (b) S. aureus, and (c) E. coli assessed using malondialdehyde as a marker for lipid peroxidation. Symbols (•) represent the mean of three technical replicates. Data are presented as scatterplots with bar ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the unirradiated control.

FIG 10

Effects of aBL on membrane permeabilization in P. aeruginosa, S. aureus, and E. coli assessed by N-phenyl-1-naphthylamine (NPN, a, b, and c) and propidium iodide (PI, d, e, and f). Symbols (•) represent the mean of three technical replicates. Data are presented as scatterplots with bar ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the negative control (C−). Positive control (C+) for membrane damage samples were treated with ethanol at 70% for 15 min.