Antimicrobial Photodynamic Treatment Research Articles

In Vitro Eradication of Planktonic, Saliva and Biofilm Bacteria Using Lingonberry Extract as a Photosensitizer for Visible Light Plus Water-Filtered Infrared-A Irradiation

Antimicrobial photodynamic treatment (aPDT) with visible light plus water-filtered infrared-A irradiation (VIS-wIRA) and natural single- or multi-component photosensitizers (PSs) was shown to have potent antimicrobial activity. The aim of this study was to obtain information on the antimicrobial effects of aPDT-VIS-wIRA with lingonberry extract (LE) against bacteria that play a role in oral health. Planktonic bacterial cultures of the Gram-positive E. faecalis T9, S. mutans DSM20523, S. oralis ATCC 35037 and S. sobrinus PSM 203513, the Gram-negative N. oralis 14F2 FG-15-7B, F. nucleatum ATCC 25586, and V. parvula DSM, the anaerobic F. nucleatum ATCC 25586 and V. parvula DSM 2008, and the total mixed bacteria from pooled saliva and supra- and subgingival plaques of volunteers were all treated and compared. aPDT-VIS-wIRA with LE as PS significantly (p < 0.008) reduced the growth of all tested Gram-positive, Gram-negative, as well as aerobic and anaerobic bacterial strains, whereas without irradiation no reductions were seen (p < 0.0001). NaCl, with or without irradiation, was ineffective. After treatment with CHX 0.2%, the highest killing rate (100%) was observed, and no bacteria (0 log10 CFU) were cultivable. The method also significantly reduced all of the bacteria present in saliva and in the gingival biofilms. Three-dimensional visualization of viable and non-viable microorganisms revealed that LE penetrated deeper into the cell wall layers than CHX 0.2%. LE was an appropriate PS for eradicating microorganisms with VIS-wIRA, either in their planktonic form or in saliva and gingival plaque biofilms. These results encourage further investigation in order to determine which LE compounds contribute to the photosensitizing effect and to evaluate the size of the effect on maintaining oral health.

Hydrogen peroxide preoxidation as a strategy for enhanced antimicrobial photodynamic action against methicillin-resistant Staphylococcus aureus.

Antimicrobial photodynamic treatment (aPDT) is a photooxidative process based on the excitation of a photosensitizer (PS) in the presence of molecular oxygen, under specific wavelengths of light. It is a promising method for advanced treatment of water and wastewater, particularly targeting disinfection challenges, such as antibiotic-resistant bacteria (ARB). Research in improved aPDT has been exploring new PS materials, and additives in general. Hydrogen peroxide (H2O2) a widely applied disinfectant, mostly in the food industry and clinical settings, present environmentally negligible residuals at the usually applied concentrations, making it friendly for the water and wastewater sectors. Here, we explored the effects of preoxidation with H2O2 followed by blue light-mediated (450 nm) aPDT using curcumin (a natural-based PS) against methicillin-resistant Staphylococcus aureus (MRSA). Results of the sequential treatment pointed to a slight hampering in aPDT efficiency at very low H2O2 concentrations, followed by an increasing cooperative effect up to a deleterious point (≥7 log10 inactivation in CFU mL-1), suggesting a synergistic interaction of preoxidation and aPDT. The increased performance in H2O2-pretreated aPDT encourages studies of optimal operational conditions for the assisted technology and describes potentials for using the described strategy to tackle the issue of ARB spread.

Quercetin mediated antimicrobial photodynamic treatment using blue light on Escherichia coli O157:H7 and Listeria monocytogenes

Interest in using an antimicrobial photodynamic treatment (aPDT) for the microbial decontamination of food has been growing. In this study, quercetin, a substance found ubiquitously in plants, was used as a novel exogenous photosensitizer with 405 nm blue light (BL) for the aPDT on foodborne pathogens, and the inactivation mechanism was elucidated. The inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in PBS solution by the quercetin and BL combination treatment reached a log reduction of 6.2 and more than 7.55 at 80 J/cm2 (68 min 21 s), respectively. When EDTA was added to investigate the reason for different resistance between two bacteria, the effect of aPDT was enhanced against E. coli O157:H7 but not L. monocytogenes. This result indicated that the lipopolysaccharide of Gram-negative bacteria operated as a protective barrier. It was experimentally demonstrated that quercetin generated the superoxide anion and hydrogen peroxide as the reactive oxygen species that oxidize and inactivate cell components. The damage to the bacterial cell membrane by aPDT was evaluated by propidium iodide, where the membrane integrity significantly (P < 0.05) decreased from 40 J/cm2 compared to control. In addition, DNA integrity of bacteria was significantly (P < 0.05) more decreased after aPDT than BL treatment. The inactivation results could be applied in liquid food industries for decontamination of foodborne pathogens, and the mechanisms data was potentially utilized for further studies about aPDT using quercetin.

Antibacterial Efficacy and Mechanisms of Curcumin-Based Photodynamic Treatment against Staphylococcus aureus and Its Application in Juices.

Antimicrobial Photodynamic Treatment (aPDT) is a non-thermal sterilization technology, which can inactivate common foodborne pathogens. In the present study, photodynamic inactivation on Staphylococcus aureus (S. aureus) with different concentrations of curcumin and light dose was evaluated and the mechanisms were also investigated. The results showed that curcumin-based aPDT could inactivate S. aureus cells by 6.9 log CFU/mL in phosphate buffered saline (PBS). Moreover, the modified Gompertz model presented a good fit at the inactivation data of S. aureus. Photodynamic treatment caused cell membrane damage as revealed by analyzing scanning electron microscopy (SEM) images. Leakage of intracellular constituents further indicated that cell membrane permeability was changed. Flow cytometry with double staining demonstrated that cell membrane integrity and the activity of nonspecific esterase were destroyed. Compared with the control group, intracellular reactive oxygen species (ROS) levels caused by photodynamic treatment significantly increased. Furthermore, curcumin-based aPDT reduced S. aureus by 5 log CFU/mL in juices. The color of the juices was also tested using a Chromatic meter, and it was found that b* values were the most markedly influenced by photodynamic treatment. Overall, curcumin-based aPDT had strong antibacterial activity against S. aureus. This approach has the potential to remove foodborne pathogens from liquid food.

Photoantimicrobials in agriculture.

Classical approaches for controlling plant pathogens may be impaired by the development of pathogen resistance to chemical pesticides and by limited availability of effective antimicrobial agents. Recent increases in consumer awareness of and/or legislation regarding environmental and human health, and the urgent need to improve food security, are driving increased demand for safer antimicrobial strategies. Therefore, there is a need for a step change in the approaches used for controlling pre- and post-harvest diseases and foodborne human pathogens. The use of light-activated antimicrobial substances for the so-called antimicrobial photodynamic treatment is known to be effective not only in a clinical context, but also for use in agriculture to control plant-pathogenic fungi and bacteria, and to eliminate foodborne human pathogens from seeds, sprouted seeds, fruits, and vegetables. Here, we take a holistic approach to review and re-evaluate recent findings on: (i) the ecology of naturally-occurring photoantimicrobials, (ii) photodynamic processes including the light-activated antimicrobial activities of some plant metabolites, and (iii) fungus-induced photosensitization of plants. The inhibitory mechanisms of both natural and synthetic light-activated substances, known as photosensitizers, are discussed in the contexts of microbial stress biology and agricultural biotechnology. Their modes-of-antimicrobial action make them neither stressors nor toxins/toxicants (with specific modes of poisonous activity), but a hybrid/combination of both. We highlight the use of photoantimicrobials for the control of plant-pathogenic fungi and quantify their potential contribution to global food security.

Photodynamic treatment of pathogens

The current viral pandemic has highlighted the compelling need for effective and versatile treatments, that can be quickly tuned to tackle new threats, and are robust against mutations. Development of such treatments is made even more urgent in view of the decreasing effectiveness of current antibiotics, that makes microbial infections the next emerging global threat. Photodynamic effect is one such method. It relies on physical processes proceeding from excited states of particular organic molecules, called photosensitizers, generated upon absorption of visible or near infrared light. The excited states of these molecules, tailored to undergo efficient intersystem crossing, interact with molecular oxygen and generate short lived reactive oxygen species (ROS), mostly singlet oxygen. These species are highly cytotoxic through non-specific oxidation reactions and constitute the basis of the treatment. In spite of the apparent simplicity of the principle, the method still has to face important challenges. For instance, the short lifetime of ROS means that the photosensitizer must reach the target within a few tens nanometers, which requires proper molecular engineering at the nanoscale level. Photoactive nanostructures thus engineered should ideally comprise a functionality that turns the system into a theranostic means, for instance, through introduction of fluorophores suitable for nanoscopy. We discuss the principles of the method and the current molecular strategies that have been and still are being explored in antimicrobial and antiviral photodynamic treatment.

Antimicrobial photodynamic treatment (aPDT) as an innovative technology to control spoilage and pathogenic microorganisms in agri-food products: An updated review

Antimicrobial photodynamic treatment (aPDT) is a light-based method developed for the inactivation of microorganisms. With the emergence of antifungal- and antibiotic-resistant microorganisms, aPDT has been suggested as a promising microbial control strategy. The treatment is based on a combination of visible light, a photosensitizer (PS), and molecular oxygen. Illuminating the PS with adequate light in the presence of oxygen will result in the generation of abundant reactive oxygen species (ROS), which further react with multiple targets within microbial cells and eventually cause cell death. The risk of resistance development is low due to the multi-target mode of action of aPDT. The efficiency of aPDT for microbial inactivation on foods can be affected by several factors such as type and characteristics of the PS, light source, food surface geometry, environmental aspects, and microbial characteristics. Although still a concept, the application of aPDT has gained rapid acceptance in many different agri-food products and associated processes, including food production, industrial processing, storage, distribution, and retail. Most of the in vitro aPDT studies have provided evidence of significant antimicrobial effects, including cases in which viability was reduced up to eight orders of magnitude. Strong antimicrobial performance of aPDT was also reported for studies involving variable food matrices, including fruit and vegetables, meat products, and milk. This review provides up-to-date information on advances in the application of aPDT in the agri-food sector, including suitable photosensitizers, sources of light, microbial inactivation efficiency, the role of food matrix composition, potentiation effects, inactivation mechanisms, and potential or prospective applications in the food industry and in other areas of agriculture. Even though the field of aPDT is advancing rapidly, future research should aim at some topics, including (1) the complete analysis of the effects of the treatment on food matrices, (2) the inactivation of currently-neglected food spoilage microorganisms, and (3) comprehensive field evaluation of aPDT for the control of plant diseases, all of which are necessary in order for the treatment to find widespread use in the agri-food sector. • aPDT is efficient at inactivating food spoilage microorganisms. • Chlorophyllin and curcumin are the natural PS of choice. • Fruit, vegetables, and meat are the most studied food matrices. • Future studies with thermophilic food spoilage organisms are needed. • Thorough food matrix analysis after treatment is still lacking.

Phenothiazinium dyes for photodynamic treatment present lower environmental risk compared to a formulation of trifloxystrobin and tebuconazole.

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents.

A Biosynthesized Near-Infrared-Responsive Nanocomposite Biomaterial for Antimicrobial and Antibiofilm Treatment.

Photodynamic inactivation (PDI) has become an appealing alternative strategy to treat infections without developing resistance to microbes. In PDI treatment, near-infrared (NIR) light is preferred because it causes less damage to normal tissues and leads to better penetration in deep tissues. Here, we develop an NIR-responsive nanomedicine for efficient broad-spectrum antimicrobial photodynamic treatment. By harnessing the biosynthetic capability of a bacterial cellulose-producing microorganism, we construct a nanocomposite biomaterial to deliver and recycle the nanomedicine. Our simple one-step biosynthetic approach does not impede the antimicrobial potency of the nanomedicine under NIR activation and requires no chemical modification. The resulting nanocomposite has been tested in antimicrobial treatment of different microorganisms, exhibiting a great potential to eliminate pathogens in biofilms and to treat in vivo infections.

Comparative study between photodynamic therapy with urucum + Led and probiotics in halitosis reduction-protocol for a controlled clinical trial.

BackgroundHalitosis is a term that defines any foul odor emanating from the oral cavity. The origin may be local or systemic. The aim of the proposed protocol is to determine whether treatment with antimicrobial photodynamic therapy (aPDT) and treatment with probiotics are effective at eliminating halitosis.Materials and methodsEighty-eight patients, from 18 to 25 years old with a diagnosis of halitosis (H2S≥112 ppb, determined by gas chromatography) will be randomly allocated to four groups (n = 22) that will receive different treatments: Group 1 –treatment with teeth brushing, dental floss and tongue scraper; Group 2 –brushing, dental floss and aPDT; Group 3 –brushing, dental floss and probiotics; Group 4 –brushing, flossing, aPDT and probiotics. The results of the halimetry will be compared before, immediately after, seven days and thirty days after treatment. The microbiological analysis of the coated tongue will be performed at these same times. The normality of the data will be determined using the Shapiro-Wilk test. Data with normal distribution will be analyzed using analysis of variance (ANOVA). Non-parametric data will be analyzed using the Kruskal-Wallis test. The Wilcoxon test will be used to analyze the results of each treatment at the different evaluation periods.Clinical trail registrationNCT03996044.

A rapid antimicrobial photodynamic water treatment strategy utilizing a xanthene dye with subsequent removal by Goethite Nanoparticles

Although widely accepted as a water sterilisation technique, chlorination results in the production of potentially harmful by-products, mainly Trihalomethanes. Furthermore, the chlorination process requires specialised infrastructure, management and high costs. In this research paper a potential alternative sterilisation technique was investigated. This rapid three-step process utilized Goethite Nanoparticles and the photosensitising capabilities of a xanthene dye. Rose Bengal (RB) a compound primarily used as a stain to diagnose damaged tissue in the eye was utilized under visible light excitation to sterilise water containing gram-positive Staphylococcus aureus and Bacillus cereus. Bacterial reductions (cfu/ml) of up to 6log10 are reported at RB concentrations of 0.5 mg/L and 10 mg/L for S. aureus and B. cereus, respectively. Goethite Nanoparticles (GNP's), an iron oxyhydroxide, were synthesised by co-precipitation of iron salts and used to adsorb RB post-sterilisation. Poly-vinyl Alcohol (PVA) functionalised GNP's were synthesised to highlight the adsorbent capabilities of the GNP surface. The adsorption capacity for uncoated GNPs was 476.19 mg/g, this reduced to 170.4 mg/g for PVA-GNP's, highlighting the highly porous nature of the synthesised GNP surface. Adsorption was optimal in slightly acidic conditions (pH5-6). The adsorption parameters best followed Lagergens Pseudo-second order kinetics with correlation coefficients close to unity. At the highest envisaged RB concentration (10 mg/L) approximately 20 mg/L GNP's was required to remove the dye from solution post-treatment. Flame Atomic Absorption analysis of the water post-removal revealed Iron concentrations of 0.058 mg/L. This correlates to removal efficacy of 99.71% with residual iron levels below the EU recommended limit of 0.2 mg/L.

Natural photosensitizers from Tripterygium wilfordii and their antimicrobial photodynamic therapeutic effects in a Caenorhabditis elegans model

Tripterygium wilfordii Hook. f. is a traditional medicinal plant and has long been used in East Asia to treat many diseases. However, the extract and active components have never been investigated as potential photosensitizers for photodynamic treatment to kill pathogenic microorganisms. Here, the antimicrobial photodynamic treatment (APDT) effects of the extract, fractions, and compounds of T. wilfordii were evaluated in vitro and in vivo. Ethanolic extract (TWE) and the photosensitizer-enriched fraction (TW-F5) were prepared from dried T. wilfordii. Six active compounds were isolated from TW-F5 by semipreparative high-performance liquid chromatography, and their chemical structures were characterized through spectroscopic and spectrometric analysis. The singlet oxygen from extracts, fractions, and compounds was measured by using the imidazole-N,N-dimethyl-4-nitrosoaniline method. These extracts, fractions, and compounds were used as photosensitizers for the inactivation of bacteria and fungi by red light at 660 nm. The in vitro APDT effects were also evaluated in the model animal Caenorhabditis elegans. APDT with TWE showed effective antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans. TW-F5, consisting of six pheophorbide compounds, also showed strong APDT activity. The photosensitizers were taken up into the bacterial cells and induced intracellular ROS production by APDT. TWE and TW-F5 also induced a strong APDT effect in vitro against skin pathogens, including Staphylococcus epidermidis and Streptococcus pyogenes. We evaluated the APDT effects of TWE and TW-F5 in C. elegans infected with various pathogens and found that PDT effectively controlled pathogenic bacteria without strong side effects. APDT reversed the growth retardation of worms induced by pathogen infection and decreased the viable pathogenic bacterial numbers associated with C. elegans. Finally, APDT with TWE increased the survivability of C. elegans infected with S. pyogenes. In summary, TWE and TW-F5 were found to be effective antimicrobial photosensitizers in PDT.

Antimicrobial Photodynamic Treatment with Mother Juices and Their Single Compounds as Photosensitizers.

The potent antimicrobial effects of antimicrobial photodynamic therapy (aPDT) with visible light plus water-filtered infrared-A irradiation and natural compounds as photosensitizers (PSs) have recently been demonstrated. The aim of this study was to obtain information on the antimicrobial effects of aPDT with mother juices against typical cariogenic oral Streptococcus pathogens in their planktonic form and determine its eradication potential on total human salivary bacteria from volunteers. Mother juices of pomegranate, bilberry, and chokeberry at different concentrations were used as PSs. The unweighted (absolute) irradiance was 200 mW cm−2, applied five minutes. Planktonic cultures of Streptococcus mutans and Streptococcus sobrinus and total mixed bacteria from pooled saliva of volunteers were treated with aPDT. Up to more than 5 log10 of S. mutans and S. sobrinus were killed by aPDT with 0.4% and 0.8% pomegranate juice, 3% and 50% chokeberry juice, and 12.5% bilberry juice (both strains). Concentrations of at least 25% (pomegranate) and >50% (chokeberry and bilberry) eradicated the mixed bacteria in saliva samples. This pilot study has shown that pomegranate mother juice is superior to the berry juices as a multicomponent PS for killing pathogenic oral bacteria with aPDT.

Inactivation kinetics of Bacillus cereus vegetative cells and spores from different sources by antimicrobial photodynamic treatment (aPDT)

Antimicrobial photodynamic treatment (aPDT) is a promising alternative to conventional thermal inactivation methods. This study aimed to evaluate Bacillus cereus vegetative cells' inactivation and spores by aPDT with new methylene blue (NMB) as photosensitizer (PS) using red light. The efficacy of aPDT was determined initially by minimal inhibitory concentration (MIC) of NMB at different concentrations and fluences. Cluster analysis from the results of MIC grouped B. cereus strains according to their aPDT susceptibility. Then, four strains (B63, B3, 436, and ATCC 14579) were selected for a survival study of aPDT with NMB in three concentrations (5, 50, and 100 μM). The viability of B. cereus vegetative cells and spores was reduced in all tested fluences. The strain ATCC 14579 (vegetative cells and spores) was the most susceptible to aPDT at relatively low fluence (25 J/cm2). The Weibull model presented a good fit for the inactivation data estimating the kinetic parameters δ (first decimal reduction) and p (shape parameter). This study contributes to the knowledge of the behavior of different strains of B. cereus regarding an emerging method of microbial inactivation. The variability of inactivation among strains will allow the development of more reliable processes.

Combined Addition of Ethanol and Ethylenediaminetetraacetic Acid Enhances Antibacterial and Antibiofilm Effects in Methylene Blue-Mediated Photodynamic Treatment against Pseudomonas aeruginosa In Vitro.

Antimicrobial photodynamic treatment (aPDT) for infection with drug-resistant bacteria has received much attention. For P.aeruginosa, however, efficient formation of biofilms and the nature of Gram-negative bacteria often limit the efficacy of aPDT. In this study, we investigated the effects of ethanol and ethylenediaminetetraacetic acid (EDTA) as additives on bacterial viability, biofilm biomass, and structures of bacteria and biofilms in methylene blue (MB)-mediated aPDT in vitro. Matured P.aeruginosa biofilms were incubated with 32-µm MB solutions with different concentrations of additives and then illuminated with 665-nm light from an LED array. The combined addition of 10% ethanol and 10mm EDTA to MB resulted in significantly greater bactericidal effects than those of MB alone and of MB with 10% ethanol or 10mm EDTA. Crystal violet assays showed significant reductions in biofilm biomass by aPDT with addition of both ethanol and EDTA compared to that in the case of aPDT with MB alone. Scanning electron microscopy showed broken bacterial cells and reduction in the cell density and amount of biofilm under those conditions. Ethanol addition alone did not improve aPDT efficacy. Reduced amount of biofilm by EDTA addition would have improved the transportation of MB and ethanol to bacteria.

Photodynamic inactivation of Botrytis cinerea by an anionic porphyrin: an alternative pest management of grapevine

Botrytis cinerea is a necrotic plant fungus that causes gray mold disease in over 200 crops, including grapevine. Due to its genetic plasticity, this fungus presents strong resistance to many fungicides. Thus, new strategies against B. cinerea are urgently needed. In this context, antimicrobial photodynamic treatment (APDT) was considered. APDT involves the use of a photosensitizer that generates reactive oxygen species upon illumination with white light. Tetra-4-sulfonatophenyl porphyrin tetra-ammonium (TPPS) was tested on B. cinerea using light. 1.5 µM TPPS completely inhibited mycelial growth. TPPS (12.5 µM) was tested on three grapevine clones from Chardonnay, Merlot and Sauvignon, grown in vitro for 2 months. Treated root apparatus of the three backgrounds increased thiol production as a molecular protection against photoactivated TPPS, leading to a normal phenotype as compared with control plantlets. Finally, 2-month-old grapevine leaves were infected with 4-day-old mycelium of B. cinerea pre-incubated or not with TPPS. The pre-treated mycelium was unable to infect the detached leaves of any of the three grapevine varieties after 72 h growth when subjected to a 16 h photoperiod, contrary to untreated mycelium. These results suggest a strong potential of photo-treatment against B. cinerea mycelium for future agricultural practices in vineyard or other cultures.

Antimicrobial photodynamic treatment as an alternative approach for Alicyclobacillus acidoterrestris inactivation

Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400–740 nm; 65–140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 μM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 μM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 μM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 μM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 μM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 μM, however at the highest concentration (50 μM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.

Chemical features of the photosensitizers new methylene blue N and S137 influence their subcellular localization and photoinactivation efficiency in Candida albicans

Antimicrobial photodynamic treatment (APDT) has emerged as an effective therapy against pathogenic fungi with both acquired and intrinsic resistance to commonly used antifungal agents. Success of APDT depends on the availability of effective photosensitizers capable of acting on different fungal structures and species. Among the phenothiazinium dyes tested as photoantifungals, new methylene blue N (NMBN) and the novel pentacyclic compound S137 are the most efficient. In the present study we compared the effects of APDT with NMBN and S137 on the survival of Candida albicans and employed a set of fluorescent probes (propidium iodide, FUN-1, JC-1, DHR-123 and DHE) together with confocal microscopy and flow cytometry to evaluate the effects of these two chemically diverse photosensitizers on cell membrane permeability, metabolism and redox status, and mitochondrial activity. Taken together, our results indicate that, due to chemical features resulting in different lipophilicity, NMBN and S137 localize to distinct subcellular structures and hence inactivate C. albicans cells via different mechanisms. S137 localizes mostly to the cell membrane and, upon light exposure, photo-oxidizes membrane lipids. NMBN readily localizes to mitochondria and exerts its photodynamic effects there, which was observed to be a less effective way to achieve cell death at lower light fluences.

Identification of carbonylated proteins in a bactericidal process induced by curcumin with blue light irradiation on imipenem-resistant Acinetobacter baumannii.

Antimicrobial photodynamic treatment is potentially an alternative to antibiotics and is also effective against viruses, fungi and some cancers. Our previous studies have shown that blue light combined with curcumin, a chemical from the turmeric plant, exerted effective antimicrobial activity via photodynamic treatment. The study reported in this paper investigates which target proteins are affected after the treatment. We treated imipenem-resistant Acinetobacter baumannii with blue light and curcumin and used protein carbonylation as a marker for oxidative damage. After treatment, the bacterial proteins were extracted and the protein carbonyls marked using dinitrophenylhydrazide. After enzyme digestion, we used liquid chromatography/nano-electrospray ionization (LC/nano-ESI) ion trap mass spectrometry to identify bacterial peptides from a customized database. The functional enrichment analyses of the identified proteins were performed using gene ontology annotation and the STRING protein-protein interaction network. The application of curcumin with blue light showed good antibacterial activity against imipenem-resistant A. baumannii. Using a shotgun proteomics approach, the carbonylated proteins in A. baumannii caused by the photolytic curcumin were identified. The results showed that the proteins related to membrane structures, translation and response to oxidative stress were preferentially modified. The photolytic curcumin treatment could be a potential alternative to antibiotics for bacterial infection. In this study, the shotgun proteomics strategy allows us to explore the possible bactericidal mechanisms under this oxidative stress. The result provides a reference for future studies on the enhancement of the action of photolytic curcumin.

Photodynamic inactivation of Candida albicans and Candida tropicalis with aluminum phthalocyanine chloride nanoemulsion

The in vitro susceptibilities of Candida albicans and Candida tropicalis to Antimicrobial Photodynamic Treatment with aluminum phthalocyanine chloride in nanoemulsion (ClAlPc/NE) were investigated. PS concentration- and fluence-dependent cell survival after APDT were compared before and after unbound extracellular PS had been washed out. The PS uptake and its subcellular localization were also determined. Exposure to light in the absence of the PS and treatment with the PS in the absence of light did not kill the fungi. APDT with ClAlPc/NE resulted in a reduction of five orders of magnitude in viability for C. albicans and between four and five orders of magnitude for C. tropicalis. Washing the cells to remove unbound PS before light exposure did not impair fungal inactivation, suggesting that cell photosensitization was mainly carried out by cell bound ClAlPc. The degree of ClAlPc uptake was dependent on its concentration. Internalization of ClAlPc by C. albicans and C. tropicalis was confirmed by confocal fluorescence microscopy that showed the PS does not penetrate the nucleus and instead accumulates in specific regions of the cytoplasm. Our results show that incorporating the water-insoluble ClAlPc into a nanoemulsion leads to an efficient formulation capable of photoinactivating both Candida species.