Antimicrobial and antibiofilm photodynamic therapy against vancomycin resistant Staphylococcus aureus (VRSA) induced infection in vitro and in vivo.

Biofilm mediated infection caused by multi-drug resistant bacteria are difficult to treat since it protects the microorganisms by host defense system, making them resistant to antibiotics and other antimicrobial agents. Combating such type of nosocomial infection, especially in immunocompromised patients, is an urgent need and foremost challenge faced by clinicians. Therefore, antimicrobial photodynamic therapy (aPDT) has been intensely pursued as an alternative therapy for bacterial infections. aPDT leads to the generation of reactive oxygen species (ROS) that destroy bacterial cells in the presence of a photosensitizer, visible light and oxygen. Here, we elucidated a possibility of its clinical application by reducing the treatment time and exposing curcumin to 20 J/cm2 of blue laser light, which corresponds to only 52 s to counteract vancomycin resistant Staphylococcus aureus (VRSA) both in vitro and in vivo. To understand the mechanism of action, the generation of total reactive oxygen species (ROS) was quantified by 2'-7'-dichlorofluorescein diacetate (DCFH-DA) and the type of phototoxicity was confirmed by fluorescence spectroscopic analysis. The data showed more production of singlet oxygen, indicating type-II phototoxicity. Different anti-biofilm assays (crystal violet and congo red assays) and microscopic studies were performed at sub-MIC concentration of curcumin followed by treatment with laser light against preformed biofilm of VRSA. The result showed significant reduction in the preformed biofilm formation. Finally, its therapeutic potential was validated in skin abrasion wistar rat model. The result showed significant inhibition of bacterial growth. Furthermore, immunomodulatory analysis with rat serum was performed. A significant reduction in expression of proinflammatory cytokines TNF-α and IL-6 were observed. Hence, we conclude that curcumin mediated aPDT with 20 J/cm2 of blue laser treatment (for 52 s) could be used against multi-drug resistant bacterial infections and preformed biofilm formation as a potential therapeutic approach.

Curcumin induced photodynamic therapy mediated suppression of quorum sensing pathway of Pseudomonas aeruginosa: An approach to inhibit biofilm in vitro.

OBJECTIVE: The objective of this study was to inhibit the Pseudomonas aeruginosa biofilm through curcumin-mediated antimicrobial photodynamic therapy (A-PDT). BACKGROUND: The mechanism behind A-PDT mediated photoinactivation depend upon reactive oxygen species (ROS) production, like singlet oxygen and free radicals. METHODS: To evaluate the antibacterial efficacy of curcumin induced A-PDT on P. aeruginosa by colony forming unit (CFU) while antibiofilm action was determined by the use of crystal violet, XTT, congored binding assay and confocal laser scanning microscope (CLSM). RESULTS: We found that curcumin with 10 J/cm2 of light reduces P. aeruginosa biofilm more efficiently than without light. Extracellular polymeric substances (EPS) production was also reduced by approx 94 % with 10 J/cm2 of light dose. CLSM images showed that the thickness of biofilms were reduced from >30 µm to <5 µm after treatment with curcumin followed by 10 J/cm2 of light irradiation. Curcumin showed better bacteriostatic activity than bactericidal activity. Singlet oxygen is primarily responsible for photodamage and cytotoxic reactions caused by curcumin-mediated APDT. Genes involved in quorum sensing (QS) pathway was also found to be inhibited after APDT. Curcumin with 5 J/cm2 light inhibits QS genes and on increasing light dose i.e10 J/cm2, we found a drastic reduction in gene expression. CONCLUSION: We conclude that the curcumin mediated A-PDT inhibits biofilm formation ofP. aeruginosa through QS pathway by the action of singlet oxygen generation which in turn reduced EPS of the biofilm.