The effects of photodynamic treatment with new methylene blue N on the Candida albicans proteome.

Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface

Inhibitory action of phenothiazinium dyes against Neospora caninum.

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9-Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019-1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9-Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9-Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control

In vitro susceptibilities of Neoscytalidium spp. sequence types to antifungal agents and antimicrobial photodynamic treatment with phenothiazinium photosensitizers

Neoscytalidium spp. are ascomycetous fungi consisting of pigmented and hyaline varieties both able to cause skin and nail infection. Their colour-based identification is inaccurate and may compromise the outcome of the studies with these fungi. The aim of this study was to genotype 32 isolates morphologically identified as Neoscytalidium dimidiatum or Neoscytalidium dimidiatum var. hyalinum by multilocus sequence typing (MLST), differentiate the two varieties by their sequence types (STs), evaluate their susceptibility to seven commercial antifungal drugs [amphotericin B (AMB), voriconazole (VOR), terbinafine (TER), 5-flucytosine (5FC), ketoconazole (KET), fluconazole (FLU), and caspofungin (CAS)], and also to the antimicrobial photodynamic treatment (APDT) with the phenothiazinium photosensitizers (PSs) methylene blue (MB), new methylene blue (NMBN), toluidine blue O (TBO), and the pentacyclic derivative S137. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, the APDT effects with each PS on the survival of ungerminated and germinated arthroconidia of both varieties were evaluated. Seven loci of Neoscytalidium spp. were sequenced on MLST revealing eight polymorphic sites and six STs. All N. dimidiatum var. hyalinum isolates were clustered in a single ST. AMB, VOR, and TER were the most effective antifungal agents against both varieties. The hyaline variety isolates were much less tolerant to the azoles than the isolates of the pigmented variety. APDT with S137 showed the lowest MIC for all the isolates of both varieties. APDT with all the PSs killed both ungerminated and germinated arthroconidia of both varieties reducing the survival up to 5 logs. Isolates of the hyaline variety were also less tolerant to APDT. APDT with the four PSs also increased the plasma membrane permeability of arthroconidia of both varieties but only NMBN and S137 caused peroxidation of the membrane lipids. © 2017 British Mycological Society

Photodynamic inactivation of conidia of the fungus Colletotrichum abscissum on Citrus sinensis plants with methylene blue under solar radiation.

Antimicrobial photodynamic treatment (APDT) is a promising light based approach to control diseases caused by plant-pathogenic fungi. In the present study, we evaluated the effects of APDT with the phenothiazinium photosensitizer methylene blue (MB) under solar radiation on the germination and viability of conidia of the pathogenic fungus Colletotricum abscissum (former Colletotrichum acutatum sensu lato). Experiments were performed both on petals and leaves of sweet orange (Citrus sinensis) in different seasons and weather conditions. Conidial suspensions were deposited on the leaves and petals surface, treated with the PS (25 or 50μM) and exposed to solar radiation for only 30min. The effects of APDT on conidia were evaluated by counting the colony forming units recovered from leaves and petals and by direct evaluating conidial germination on the surface of these plant organs after the treatment. To better understand the mechanistic of conidial photodynamic inactivation, the effect of APDT on the permeability of the conidial plasma membrane was assessed using the fluorescent probe propidium iodide (PI) together with flow cytometry and fluorescence microscopy. APDT with MB and solar exposure killed C. abscissum conidia and prevented their germination on both leaves and petals of citrus. Reduction of conidial viability was up to three orders of magnitude and a complete photodynamic inactivation was achieved in some of the treatments. APDT damaged the conidial plasma membrane and increased its permeability to PI. No damage to sweet orange flowers or leaves was observed after APDT. The demonstration of the efficacy of APDT on the plant host represents a further step towards the use of the method for control phytopathogens in the field

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

In vitro and in vivo photodynamic efficacies of novel and conventional phenothiazinium photosensitizers against multidrug-resistant Candida auris

The fast-emerging and multidrug-resistant Candida auris is the first fungal pathogen to be considered a threat to global public health. Thus, there is a high unmet medical need to develop new therapeutic strategies to control this species. Antimicrobial photodynamic therapy (APDT) is a promising alternative that simultaneously targets and damages numerous microbial biomolecules. Here, we investigated the in vitro and in vivo effects of APDT with four phenothiazinium photosensitizers: (i) methylene blue (MB), (ii) toluidine blue (TBO), and two MB derivatives, (iii) new methylene blue (NMBN) and (iv) the pentacyclic derivative S137, against C.auris. To measure the in vitro efficacy of each PS, minimal inhibitory concentrations (MICs) and survival fraction were determined. Also, the efficiency of APDT was evaluated in vivo with the Galleria mellonella insect model for infection and treatment. Although the C. auris strain used in our study was shown to be resistant to the most-commonly used clinical antifungals, it could not withstand the damages imposed by APDT with any of the four photosensitizers. However, for the in vivo model, only APDT performed with S137 allowed survival of infected G.mellonella larvae. Our results show that structural and chemical properties of the photosensitizers play a major role on the outcomes of in vivo APDT and underscore the need to synthesize and develop novel photosensitizing molecules against multidrug-resistant microorganisms. Graphical abstract: [Figure not available: see fulltext.]

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