Effects of antimicrobial photodynamic therapy with photodithazine® on methicillin-resistant Staphylococcus aureus (MRSA): Studies in biofilms and experimental model with Galleria mellonella.

Staphylococcus aureus infections are a severe health problem due to the high mortality rate. Conventional treatment of these infections is via the administration of antibiotics. However, its indiscriminate use can select resistant microorganisms. Thus, it is necessary to develop alternatives for antibiotic therapy. Antimicrobial Photodynamic Therapy (aPDT), a therapeutic method that associates a photosensitizer (PS), a light source with adequate wavelength to the PS, interacts with molecular oxygen generating reactive oxygen species responsible for cell inactivation, is a viable alternative. This work aimed to analyze, in vitro and in vivo, the action of aPDT with PS Photodithazine® (PDZ) on the methicillin-resistant S. aureus (MRSA) strain. In the in vitro method, the S. aureus biofilm was incubated with PDZ at 50 and 75 µg.mL-1 for 15 min, adopting the light dose of 25, 50, and 100 J/cm2. In addition, PS interaction, formation of reactive oxygen species (ROS), bacterial metabolism, adhesion, bacterial viability, and biofilm structure were evaluated by scanning electron microscopy. Subsequently, the strain was inoculated into models of Galleria mellonella, and the survival curve, health scale, blood cell analysis, and CFU/mL of S. aureus in the hemolymph were analyzed after aPDT. In the in vitro results, bacterial reduction was observed in the different PDZ concentrations, highlighting the parameters of 75 µg.mL-1 of PDZ and 100 J/cm2. As for in vivo results, aPDT increased survival and stimulated the immune system of G. mellonella infected by S. aureus. aPDT proved effective in both models, demonstrating its potential as an alternative therapy in treating MRSA bacterial infections.

Antimicrobial photodynamic therapy with curcumin on methicillin-resistant Staphylococcus aureus biofilm.

Healthcare-Associated Infections (HAI) effect approximately 1.5 million individuals worldwide. Among the causes of HAIs in Latin America, Staphylococcus aureus presents a severe danger due to its rapid spread and ease of developing antibiotic resistance. Upon acquiring methicillin resistance, it receives the classification Methicillin-Resistant Staphylococcus aureus (MRSA), responsible for 40 to 60% of HAIs. The increase in resistant microorganisms led to the search for alternative methods, such as antimicrobial Photodynamic Therapy (aPDT), forming Reactive Oxygen Species (ROS), leading bacterial cells to death. The objective of this work was to evaluate in vitro the antimicrobial action of PDT with curcumin in MRSA biofilm. The strains were induced to form biofilm and incubated with curcumin for 20 min, irradiated with LED (Light Emitting Diode) 450 nm, at 110 mW/cm2, 50 J/cm2 for 455 s, subsequently counting the Colony Forming Units, Scanning Electron Microscopy (SEM) micrographs, Confocal Microscopy images, Resazurin dye test, ROS quantification to assess the effect of PDT on biofilm. The results show that PDT with curcumin reduced the biofilm growth of the MRSA strain. In addition, confocal microscopy showed that curcumin was internalized by S. aureus in the cells at the concentration used, and when isolated, curcumin and the irradiation parameter did not show cytotoxicity. The study demonstrated that the PDT in the established parameters reduced the growth of the MRSA strain biofilm, making it a relevant alternative possibility for the inactivation of this strain.

Efficiency of Antimicrobial Photodynamic Therapy with Photodithazine® on MSSA and MRSA Strains.

Staphylococccus aureus is a ubiquitous and opportunistic bacteria associated with high mortality rates. Antimicrobial photodynamic therapy (aPDT) is based on the application of a light source and a photosensitizer that can interact with molecular oxygen, forming Reactive Oxygen Species (ROS) that result in bacterial inactivation. This study aimed to analyze, in vitro, the action of aPDT with Photodithazine® (PDZ) in methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains. The strains were incubated with PDZ at 25, 50, 75, and 100 mg/L for 15 min and irradiated with fluences of 25, 50, and 100 J/cm2. The internalization of PDZ was evaluated by confocal microscopy, the bacterial growth by counting the number of colony-forming units, as well as the bacterial metabolic activity post-aPDT and the production of ROS. In both strains, the photosensitizer was internalized; the production of ROS increased when the aPDT was applied; there was a bacterial reduction compared to the control at all the evaluated fluences and concentrations; and, in most parameters, it was obtained complete inactivation with significant difference (p < 0.05). The implementation of aPDT with PDZ in clinical strains of S. aureus has resulted in its complete inactivation, including the MRSA strains.

Characterization and antimicrobial effect of a bioinspired thymol coating formed on titanium surface by one-step immersion treatment.

OBJECTIVE: To develop an antimicrobial and anti-adherent thymol (TOH)-containing coating on titanium (Ti) by a bioinspired one-step biocompatible method. METHODS: A nanolayer of adsorbed TOH (TOH-NL-Ti) was formed by an easy deep coating method on Ti surface. The treatment consists in a simple one-step immersion process in a TOH-containing solution. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), potentiodynamic electrochemical technique, open circuit potential records, Atomic Force Microscopy (AFM) and measurements of TOH release were used to characterize TOH-NL-Ti. Live/Dead staining and plate counting were employed to quantify attached and living adhered bacteria, respectively. Biocompatibility and cytotoxicity in fibroblastic and pre-osteoblastic cell lines were evaluated by acridine orange staining and MTT assay, respectively. RESULTS: TOH adsorbed on TOH-NL-Ti was detected by ATR-FTIR and electrochemical techniques. ATR-FTIR results showed that TOH nanofilms development involves spontaneous production of ketonic structures on Ti surface. AFM analysis revealed that the thickness of the TOH-NL was below 80 nm. Finally, microbiological assays confirmed that TOH-NL-Ti can inhibit the adhesion and kill attached bacteria leading to the eradication of leaving cells on its surface. After 24 h of biocidal release, the antimicrobial effect is also significant (a decrease of 3 orders in the number of attached bacteria). SIGNIFICANCE: The formation of TOH-NL-Ti nanolayer is a simple strategy able to be applied by not specially trained personnel, to reduce implant infection risks, ensure highly effective antimicrobial action and inhibition of bacterial adhesion on Ti surfaces without showing toxic effects for pre-osteoblastic and fibroblastic cells.