Faina Nakonechny

Intracellular Antimicrobial Photodynamic Therapy: A Novel Technique for Efficient Eradication of Pathogenic Bacteria

The increasing resistance of bacteria to antibiotics is a serious problem, caused in part by excessive and improper use of these drugs. One alternative to traditional antibiotic therapy is photodynamic antimicrobial chemotherapy (PACT) which is based on the use of a photosensitizer (PS), activated by illumination with visible light. The poor penetration of visible light through the skin limits the application of PACT to the treatment of skin infections or the use of invasive procedures. To overcome this problem we report the exploitation of light emitted as a result of the chemiluminescent reaction of luminol to excite the PS and we call this process chemiluminescent photodynamic antimicrobial therapy (CPAT). We studied the effect of free and liposome‐encapsulated PS (methylene blue or toluidine blue) on bacteria under excitation by either white external light or chemiluminescence emitted by free or liposome‐enclosed luminol. PACT showed slightly better performance that CPAT for free and encapsulated PS for both types of bacteria. CPAT resulted in a three log suppression of Staphylococcus aureus and two log suppression of Escherichia coli growth. The use of CPAT may prove to be a novel and more effective form of antimicrobial therapy, particularly for internal infections not easily accessible to traditional PACT.

Photodynamic antimicrobial chemotherapy by liposome-encapsulated water-soluble photosensitizers

Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensitizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gram-negative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.

Sonodynamic Excitation of Rose Bengal for Eradication of Gram-Positive and Gram-Negative Bacteria

Photodynamic antimicrobial chemotherapy based on photosensitizers activated by illumination is limited by poor penetration of visible light through skin and tissues. In order to overcome this problem, Rose Bengal was excited in the dark by 28 kHz ultrasound and was applied for inactivation of bacteria. It is demonstrated, for the first time, that the sonodynamic technique is effective for eradication of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The net sonodynamic effect was calculated as a 3-4 log10 reduction in bacteria concentration, depending on the cell and the Rose Bengal concentration and the treatment time. Sonodynamic treatment may become a novel and effective form of antimicrobial therapy and can be used for low-temperature sterilization of medical instruments and surgical accessories.

Eradication of Gram‐Positive and Gram‐Negative Bacteria by Photosensitizers Immobilized in Polystyrene

Immobilization of photosensitizers in polymers opens prospects for their continuous and reusable application. Methylene blue (MB) and Rose Bengal were immobilized in polystyrene by mixing solutions of the photosensitizers in chloroform with a polymer solution, followed by air evaporation of the solvent. This procedure yielded 15–140 μm polymer films with a porous surface structure. The method chosen for immobilization ensured 99% enclosure of the photosensitizer in the polymer. The antimicrobial activity of the immobilized photosensitizers was tested against Gram‐positive and Gram‐negative bacteria. It was found that both immobilized photosensitizers exhibited high antimicrobial properties, and caused by a 1.5–3 log10 reduction in the bacterial concentrations to their total eradication. The bactericidal effect of the immobilized photosensitizers depended on the cell concentration and on the illumination conditions. Scanning electron microscopy was used to prove that immobilized photosensitizers excited by white light caused irreversible damage to microbial cells. Photosensitizers immobilized on a solid phase can be applied for continuous disinfection of wastewater bacteria.

Polymer-Immobilized Photosensitizers for Continuous Eradication of Bacteria

The photosensitizers Rose Bengal (RB) and methylene blue (MB), when immobilized in polystyrene, were found to exhibit high antibacterial activity in a continuous regime. The photosensitizers were immobilized by dissolution in chloroform, together with polystyrene, with further evaporation of the solvent, yielding thin polymeric films. Shallow reservoirs, bottom-covered with these films, were used for constructing continuous-flow photoreactors for the eradication of Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and wastewater bacteria under illumination with visible white light using a luminescent lamp at a 1.8 mW·cm−2 fluence rate. The bacterial concentration decreased by two to five orders of magnitude in separate reactors with either immobilized RB or MB, as well as in three reactors connected in series, which contained one of the photosensitizers. Bacterial eradication reached more than five orders of magnitude in two reactors connected in series, where the first reactor contained immobilized RB and the second contained immobilized MB.

Increased copper bioremediation ability of new transgenic and adapted Saccharomyces cerevisiae strains

Environmental pollution with heavy metals is a very serious ecological problem, which can be solved by bioremediation of metal ions by microorganisms. Yeast cells, especially Saccharomyces cerevisiae, are known to exhibit a good natural ability to remove heavy metal ions from an aqueous phase. In the present work, an attempt was made to increase the copper-binding properties of S. cerevisiae. For this purpose, new strains of S. cerevisiae were produced by construction and integration of recombinant human MT2 and GFP-hMT2 genes into yeast cells. The ySA4001 strain expressed GFP-hMT2p under the constitutive pADH1 promoter and the ySA4002 and ySA4003 strains expressed hMT2 and GFP-hMT2 under the inducible pCUP1 promoter. An additional yMNWTA01 strain was obtained by adaptation of the BY4743 wild type S. cerevisiae strain to high copper concentrations. The yMNWTA01, ySA4002, and ySA4003 strains exhibited an enhanced ability for copper ion bioremediation.

Olive Oil-Based Delivery of Photosensitizers for Bacterial Eradication

Olive oil is a natural product of Olea europaea. It contains triacylglycerols of unsaturated and saturated fatty acids as well as free acids and numerous other biologically active components. Modern pharmaceutical industries are turning to natural herbal sources in order to find effective, low allergenic and non-irritating components that can be used in drug delivery systems or as recipients for both hydrophobic and hydrophilic active agents. Combining hydrophobic compounds with olive oil components is not problematic at all. However, this is quite different for hydrophilic compounds. One possible way for overcoming this problem is by mechanochemical treatment. This method has become widespread for preparing powdered solid materials in a large variety of compositions and involves the use of a conventional high-energy ball mill to initiate chemical reactions and structural changes of materials in solid-phase processes. Mechanochemical activation appears to be an environmentally friendly method, since it does not require organic solvents (Grigorieva et al., 2004; Margetic, 2005; Lugovskoy et al., 2008; Lugovskoy et al., 2009). It was shown that the mechanochemical method enabled some olive oil components to covalently attach to talc or to titanium dioxide the solid ingredients of creams, ointments and powders (Nisnevitch et al., 2011). The remaining components were deeply absorbed by solid phases. New solid-phase composite materials which combined useful properties of various components with a different nature were thus created. Talc combined with olive oil exhibited good antioxidant properties scavenging ca. 40% of free radicals. Olive oil phenols with one or two hydroxyl groups, such as hydroxytyrosol, caffeic acid, photocatechuic acid, syringic acid, derivatives of elenolic acid, derivatives of oleuropein, tyrosol and some others are among the olive oil components responsible for its in vitro antioxidative activity (Papadopoulos & Boskou 1991; Briante et al., 2001; Lesage-Meessen et al., 2001; Tovar et al., 2001; Vissers et al., 2004). These compounds retain their antioxidant properties when combined with talc by a mechanochemical method. Furthermore, the possibility of combining water-soluble ascorbic acid (vitamin C) with olive oil on a talc or titanium dioxide support using mechanochemical activation has been reported (Nisnevitch et al., 2011). These triple mixtures (support-olive oil-ascorbic acid) scavenged free radicals instantly and totally due to the presence of ascorbic acid, which is a well-known effective

Antibacterial Properties of Rose Bengal Immobilized in Polymer Supports

Photosensitizers immobilized in polymers can serve as antibacterial surfaces or coatings and can be applied for disinfection of water or medical instruments. The antibacterial activity of the immobilized photosensitizers is based on their excitation by visible light followed by energy transfer from the photosensitizers to oxygen dissolved in an aqueous phase which produces reactive oxygen species that cause irreversible damage to bacterial cells. The photosensitizer Rose Bengal immobilized in polystyrene, polycarbonate and poly (methyl methacrylate) was shown to eradicate Gram-positive Staphylococcus aureus bacteria under moderate illumination.