Antimicrobial Photodynamic Therapy Mediated by Fotenticine and Methylene Blue on Planktonic Growth, Biofilms, and Burn Infections of Acinetobacter baumannii

Abstract

Antimicrobial photodynamic therapy (aPDT) is considered a promising alternative strategy to control Acinetobacter baumannii infections. In this study, we evaluated the action of aPDT mediated by a new photosensitizer derivative from chlorin e-6 (Fotoenticine-FTC) on A. baumannii, comparing its effects with methylene blue (MB). For this, aPDT was applied on A. baumannii in planktonic growth, biofilms, and burn infections in Galleria mellonella. The absorption of FTC and MB by bacterial cells was also evaluated using microscopic and spectrophotometric analysis. The results of planktonic cultures showed that aPDT reduced the number of viable cells compared to the non-treated group for the reference and multidrug-resistant A. baumannii strains. These reductions varied from 1.4 to 2 log₁₀ CFU for FTC and from 2 log₁₀ CFU to total inhibition for MB. In biofilms, aPDT with MB reduced 3.9 log₁₀ CFU of A. baumannii, whereas FTC had no effect on the cell counts. In G. mellonella, only MB-mediated aPDT had antimicrobial activity on burn injuries, increasing the larvae survival by 35%. Both photosensitizers were internalized by bacterial cells, but MB showed a higher absorption compared to FTC. In conclusion, MB had greater efficacy than FTC as a photosensitizer in aPDT against A. baumannii.

Keywords: Acinetobacter baumannii; Galleria mellonella; burns; chlorin; fotoenticine; methylene blue; photodynamic therapy.

Figure 1

Means and standard deviation of viable cells of A. baumannii (log₁₀ CFU) in planktonic growth for the following groups: absence of photosensitizer and light (P-L-), FTC without light (FTC+L-), MB without light (MB+L-), absence of photosensitizer with light (P-L+), FTC with light (FTC+L+), and MB with light (MB+L+). (a) A. baumannii reference strain (ATCC 19606). (b) A. baumannii A2 clinical strain. (c) A. baumannii A4 clinical strain. ANOVA and Tukey test: different letters represent statistical difference between the groups (p ≤ 0.05).

Figure 2

Means and standard deviation of viable cells of A. baumannii (log₁₀ CFU) in biofilms for the following groups: absence of photosensitizer and light (P-L-), FTC without light (FTC+L-), MB without light (MB+L-), absence of photosensitizer with light (P-L+), FTC with light (FTC+L+), and MB with light (MB+L+). (a) Experiment performed to test the photosensitizer MB. (b) Experiment performed to test the photosensitizer FTC. ANOVA and Tukey test: different letters represent statistical difference between the groups (p ≤ 0.05).

Figure 3

Burn infection model in G. mellonella. Larvae were injured with a heated steel instrument in their cuticle (a), resulting in a burn lesion (b). After larvae were locally infected with a suspension of A. baumannii (c), it was possible to observe the progression of systemic infection (d).

Figure 4

Survival curve of G. mellonella obtained in the following groups: healthy larvae not treated, healthy larvae treated with light alone, larvae with burn infected by A. baumannii and not treated, larvae with burn infected by A. baumannii and treated with MB-mediated aPDT, and larvae with burn infected by A. baumannii and treated with FTC-mediated aPDT. Comparison of survival curves by Log-rank test: no statistically significant differences were found for the groups “burn infection + aPDT with MB” (p = 0.1827) and “burn infection + aPDT with FTC” (p = 0.5780) in relation to “burn infection not treated”.

Figure 5

Means of scores obtained in the health index analysis for the groups: healthy larvae not treated (control group), burn infection not treated group, burn infection + aPDT with MB group, and burn infection + aPDT with FTC group. ANOVA and Tukey test (p ≤ 0.05). (a) Movement activity: burn infection not treated group compared to burn infection + aPDT with MB group (p = 0.2668) and burn infection + aPDT with FTC group (p = 0.2321). (b) Cocoon formation: burn infection not treated group compared to burn infection + aPDT with MB group (p = 0.8055) and burn infection + aPDT with FTC group (p = 0.9989). (c) Melanization: burn infection not treated group compared to burn infection + aPDT with MB group (p = 0.1713) and burn infection + aPDT with FTC group (p = 0.6183).

Figure 6

Confocal microscopy of MB and FTC internalization by A. baumannii planktonic cells. (a,d) A. baumannii cells with DAPI stained; Internalization of MB (b) and FTC (e) by A. baumannii cells; The two overlapping images for MB (c) and FCT (f).

Figure 7

Means and SD of absorbance values (600 nm) in optical density values showing the absorption of MB and FTC by the A. baumannii cells (ATCC 19606). (Water) A. baumannii in sterile distilled water only; (MB) A. baumannii treated with MB; (FTC) A. baumannii treated with FTC.