Search

We investigated the in vitro antibacterial activity of the combination rifampicin (RIF) + polymyxin B (PB) against extensively drug-resistant (XDR) Klebsiella pneumoniae isolates. We evaluated clinical isolates co-resistant to PB (non-mcr carriers; eptB, mgrB, pmr operon, and ramA mutations) and to carbapenems (KPC, CTX-M, and SHV producers; including KPC + NDM co-producer), belonging to sequence types (ST) ST16, ST11, ST258, ST340, and ST437. We used the standard broth microdilution method to determine RIF and PB minimum inhibitory concentration (MIC) and the checkerboard assay to evaluate the fractional inhibitory concentration index (FICI) of RIF + PB as well as to investigate the lowest concentrations of RIF and PB that combined (RIF + PB) had antibacterial activity. Time-kill assays were performed to evaluate the synergistic effect of the combination against selected isolates. PB MIC (32-256 µg/mL) and RIF MIC (32-1024 µg/mL) were determined. FICI (<0.5) indicated a synergistic effect for all isolates evaluated for the combination RIF + PB. Our results showed that low concentrations of PB (PB minimal effective antibiotic concentration [MEAC], ≤0.25-1 µg/mL) favor RIF (≤0.03-0.125 µg/mL) to reach the bacterial target and exert antibacterial activity against PB-resistant isolates, and the synergistic effect was also observed in time-kill results. The combination of RIF + PB showed in vitro antibacterial activity against XDR, carbapenem-, and PB-resistant K. pneumoniae and could be further studied as a potential combination therapy, with cost-effectiveness and promising efficacy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Assessing the in vitro toxicity of compounds on cell cultures is an important step during the screening of candidate molecules for diverse applications. Among the strategies employed to determine cytotoxicity, MTT, neutral red, and resazurin are commonly used. Methylene blue (MB), a phenothiazinium salt, has several uses, such as dye, redox indicator, and even as treatment for human disease and health conditions, such as malaria and methemoglobinemia. However, MB has only been sparsely used as a cellular toxicity indicator. As a viability indicator, MB is mostly applied to fixed cultures at high concentrations, especially when compared to MTT or neutral red. Here we show that MB and its related compounds new methylene blue (NMB), toluidine blue O (TBO), and dimethylmethylene blue (DMMB) can be used as cytotoxicity indicators in live (non-fixed) cells treated for 72 h with DMSO and cisplatin. We compared dye uptake between phenothiazinium dyes and neutral red by analyzing supernatant and cell content via visible spectra scanning and microscopy. All dyes showed a similar ability to assess cell toxicity compared to either MTT or neutral red. Our method represents a cost-effective alternative to in vitro cytotoxicity assays using cisplatin or DMSO, indicating the potential of phenothiazinium dyes for the screening of candidate drugs and other applications., (© 2023. The Author(s).)

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., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The emergence of opportunistic pathogens and the selection of resistant strains have created a grim scenario for conventional antimicrobials. Consequently, there is an ongoing search for alternative techniques to control these microorganisms. One such technique is antimicrobial photodynamic therapy (aPDT), which combines photosensitizers, light, and molecular oxygen to produce reactive oxygen species and kill the target pathogen. Here, the in vitro susceptibilities of three fungal pathogens, namely Candida albicans, Aspergillus nidulans, and Colletotrichum abscissum to aPDT with zinc(II) phthalocyanine (ZnPc) derivative complexes were investigated. Three ZnPc bearing thiopyridinium substituents were synthesized and characterized by several spectroscopic techniques. The Q-band showed sensitivity to the substituent with high absorptivity coefficient in the 680-720 nm region. Derivatization and position of the rings with thiopyridinium units led to high antifungal efficiency of the cationic phthalocyanines, which could be correlated with singlet oxygen quantum yield, subcellular localization, and cellular uptake. The minimum inhibitory concentrations (MIC) of the investigated ZnPc-R complexes against the studied microorganisms were 2.5 μM (C. albicans) and 5 μM (A. nidulans and C. abscissum). One ZnPc derivative achieved complete photokilling of C. albicans and, furthermore, yielded low MIC values when used against the tolerant plant-pathogen C. abscissum. Our results show that chemical modification is an important step in producing better photosensitizers for aPDT against fungal pathogens., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This study aimed to assess the ultrapure cannabidiol (CBD) antibacterial activity and to investigate the antibacterial activity of the combination CBD + polymyxin B (PB) against Gram-negative (GN) bacteria, including PB-resistant Gram-negative bacilli (GNB). We used the standard broth microdilution method, checkerboard assay, and time-kill assay. CBD exhibited antibacterial activity against Gram-positive bacteria, lipooligosaccharide (LOS)-expressing GN diplococcus (GND) (Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis), and Mycobacterium tuberculosis, but not against GNB. For most of the GNB studied, our results showed that low concentrations of PB (≤ 2 µg/mL) allow CBD (≤ 4 µg/mL) to exert antibacterial activity against GNB (e.g., Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii), including PB-resistant GNB. CBD + PB also showed additive and/or synergistic effect against LOS-expressing GND. Time-kill assays results showed that the combination CBD + PB leads to a greater reduction in the number of colony forming units per milliliter compared to CBD and PB alone, at the same concentration used in combination, and the combination CBD + PB was synergistic for all four PB-resistant K. pneumoniae isolates evaluated. Our results show that CBD has translational potential and should be further explored as a repurposed antibacterial agent in clinical trials. The antibacterial efficacy of the combination CBD + PB against multidrug-resistant and extensively drug-resistant GNB, especially PB-resistant K. pneumoniae, is particularly promising., (© 2022. The Author(s).)

Metarhizium fungi are soil-inhabiting ascomycetes which are saprotrophs, symbionts of plants, pathogens of insects, and participate in other trophic/ecological interactions, thereby performing multiple essential ecosystem services. Metarhizium species are used to control insect pests of crop plants and insects that act as vectors of human and animal diseases. To fulfil their functions in the environment and as biocontrol agents, these fungi must endure cellular stresses imposed by the environment, one of the most potent of which is solar ultraviolet (UV) radiation. Here, we examine the cellular stress biology of Metarhizium species in context of their photobiology, showing how photobiology facilitates key aspects of their ecology as keystone microbes and as mycoinsectides. The biophysical basis of UV-induced damage to Metarhizium, and mechanistic basis of molecular and cellular responses to effect damage repair, are discussed and interpreted in relation to the solar radiation received on Earth. We analyse the interplay between UV and visible light and how the latter increases cellular tolerance to the former via expression of a photolyase gene. By integrating current knowledge, we propose the mechanism through which Metarhizium species use the visible fraction of (low-UV) early-morning light to mitigate potentially lethal damage from intense UV radiation later in the day. We also show how this mechanism could increase Metarhizium environmental persistence and improve its bioinsecticide performance. We discuss the finding that visible light modulates stress biology in the context of further work needed on Metarhizium ecology in natural and agricultural ecosystems, and as keystone microbes that provide essential services within Earth's biosphere., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Metarhizium is an important genus of soil-inhabiting fungi that are used for the biological control of insects. The efficiency of biocontrol is dependent on the maintenance of inoculum viability under adverse field conditions such as solar ultraviolet (UV) radiation. Therefore, increasing the tolerance of Metarhizium to UV radiation is necessary. It was previously established that, in mycelium, exposure to visible light increases tolerance to UV radiation. Similarly, growth under visible light for 14 days induces the production of tolerant conidia. However, a study evaluating if and how visible light affects conidia and their relationship with UV radiation was never performed. Here, we report that a relatively short and timed exposure to light around the time of conidiation is sufficient to induce the production of conidia with increased photoreactivating capacity and UV tolerance in Metarhizium acridum. Conidia produced by this method retain their characteristic higher tolerance even after many days of being transferred to the dark. Furthermore, we show that mature conidia of M. acridum and Metarhizium brunneum can still answer to light and regulate UV tolerance, suggesting that gene expression is possible even in dormant spores. Being able to respond to light in the dormant stages of development is certainly an advantage conferring improved environmental persistence to Metarhizium., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH 2 PO 4 , pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (K m(GSSG) :0.10 ± 0.02 mM; k cat(GSSG) :0.076 ± 0.003 s -1 ; K m(NADPH) :0.006 ± 0.001 mM; k cat(NADPH) : 0.080 ± 0.003 s -1 ). The IC 50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 μM, 11 ± 2 μM, 0.7 ± 0.1 μM, and 0.9 ± 0.2 μM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antimicrobial photodynamic therapy (aPDT) is a complementary therapeutic modality for periodontal and endodontic diseases, in which Gram-negative bacteria are directly involved. Currently, there are few evidences regarding the effects of aPDT on bacterial components such as lipopolysaccharide (LPS) and it would represent a major step forward in the clinical use of this therapy. In this context, this study aimed to evaluate the efficacy of different photosensitizers (PSs) used in aPDT in LPS inhibition. Four PSs were used in this study: methylene blue (MB), toluidine blue (TBO), new methylene blue (NMB), and curcumin (CUR). Different approaches to evaluate LPS interaction with PSs were used, such as spectrophotometry, Limulus amebocyte lysate (LAL) test, functional assays using mouse macrophages, and an in vivo model of LPS injection. Spectrophotometry showed that LPS decreased the absorbance of all PSs used, indicating interactions between the two species. LAL assay revealed significant differences in LPS concentrations upon pre-incubation with the different PSs. Interestingly, the inflammatory potential of LPS decreased after previous treatment with the four PSs, resulting in decreased secretion of inflammatory cytokines by macrophages. In vivo, pre-incubating curcumin with LPS prevented animals from undergoing septic shock within the established time. Using relevant models to study the inflammatory activity of LPS, we found that all PSs used in this work decreased LPS-induced inflammation, with a more striking effect observed for NMB and curcumin. These data advance the understanding of the mechanisms of LPS inhibition by PSs.

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/cm 2 ). 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/cm 2 ). 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/cm 2 ). 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 (10 5 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/cm 2 ). 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/cm 2 ). 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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Photodynamic inactivation of bacterial and fungal pathogens is a promising alternative to the extensive use of conventional single-target antibiotics and antifungal agents. The combination of photosensitizers and adjuvants can improve the photodynamic inactivation efficiency. In this regard, it has been shown that the use of potassium iodide (KI) as adjuvant increases pathogen killing. Following our interest in this topic, we performed the co-encapsulation of a neutral porphyrin photosensitizer (designated as P1) and KI into micelles and tested the obtained nanoformulations against the human pathogenic fungus Candida albicans. The results of this study showed that the micelles containing P1 and KI displayed a better photodynamic performance towards C. albicans than P1 and KI in solution. It is noteworthy that higher concentrations of KI within the micelles resulted in increased killing of C. albicans. Subcellular localization studies by confocal fluorescence microscopy revealed that P1 was localized in the cell cytoplasm, but not in the nuclei or mitochondria. Overall, our results show that a nanoformulation containing a photosensitizer plus an adjuvant is a promising approach for increasing the efficiency of photodynamic treatment. Actually, the use of this strategy allows a considerable decrease in the amount of both photosensitizer and adjuvant required to achieve pathogen killing.

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.

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., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Light is an important stimulus for fungi as it regulates many diverse and important biological processes. Metarhizium acridum is an entomopathogenic fungus currently used for the biological control of insect pests. The success of this approach is heavily dependent on tolerance to environmental stresses. It was previously reported that light exposure increases tolerance to ultraviolet radiation in M. acridum There is no information in the literature about how light globally influences gene expression in this fungus. We employed a combination of mRNA-Sequencing and high-throughput proteomics to study how light regulates gene expression both transcriptionally and post-transcriptionally. Mycelium was exposed to light for 5 min and changes at the mRNA and protein levels were followed in time-course experiments for two and four hours, respectively. After light exposure, changes in mRNA abundance were observed for as much as 1128 genes or 11.3% of the genome. However, only 57 proteins changed in abundance and at least 347 significant changes at the mRNA level were not translated to the protein level. We observed that light downregulated subunits of the eukaryotic translation initiation factor 3, the eIF5A-activating enzyme deoxyhypusine hydroxylase, and ribosomal proteins. We hypothesize that light is perceived as a stress by the cell that responds to it by reducing translational activity. Overall, our results indicate that light acts both as a signal and a stressor to M. acridum and highlight the importance of measuring protein levels in order to fully understand light responses in fungi., (Copyright © 2019 Brancini et al.)

Fusarium keratoplasticum and Fusarium moniliforme are filamentous fungi common in the environment and cause mycosis in both animals and plants. Human infections include mycetoma, keratitis and onychomycosis, while deeper mycosis occurs in immunocompromised patients. Most of the Fusarium spp. are frequently resistant to treatment with currently used antifungals. The frequent occurrence of antifungal resistance has motivated the study of antimicrobial photodynamic therapy as an alternative treatment for fungal infections. Many studies have investigated the in vitro use of antimicrobial photodynamic therapy to kill fungi, but rarely in animal models of infection. Thus, here we employed the invertebrate wax moth Galleria mellonella to study the in vivo effects of antimicrobial photodynamic therapy with three different phenothiazinium photosensitizers, methylene blue, new methylene blue N and the pentacyclic S137 against infection with microconidia of Fusarium keratoplasticum and Fusarium moniliforme. The effect of antimicrobial photodynamic therapy using these photosensitizers and light-emitting diodes with an emission peak at 635 nm and an integrated irradiance from 570 to 670 nm of 9.8 mW cm -2 was investigated regarding the toxicity, fungal burden, larval survival and cellular immune response. The results from this model indicate that antimicrobial photodynamic therapy with methylene blue, new methylene blue N and S137 is efficient for the treatment of infection with F. keratoplasticum and F. moniliforme. The efficiency can be attributed to the fungal cell damage caused by antimicrobial photodynamic therapy which facilitates the action of the host immune response., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The variability in spore survival during spray drying of 12 Bacillus cereus strains was evaluated. B. cereus spores were inoculated on whole milk (7.2 ± 0.2 log 10  spores/g dry weight) and processed in a spray-dryer. Twelve independent experiments were carried out in triplicate. The spore count was determined before and after each drying process, based on the dry weight of whole milk and powdered milk. Then, the number of decimal reductions (γ) caused by the spray drying process was calculated. B. cereus strains presented γ values ranging from 1.0 to 4.7 log10 spores/g dry weight, with a high coefficient of variation (CV) of 46.1%. Cluster analysis allowed to group B. cereus as sensitive (strains 511, 512, 540, 432 and ATCC 14579), intermediate (strains B18, B63, and B86) and resistant strains (strains B3, B94, B51 and 436). Three strains (one of each group) were selected for further investigation and characterization of their physicochemical and molecular (proteomics) differences. Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) were used to determine physicochemical characteristics and glass transition temperature (T g ), respectively. No differences in signs among the three strains were found in spectra ranging from 900 to 4000 cm -1 . The endothermic peak ranged between 54 and 130 °C for strain 540; between 81 and 163 °C for strain B63; and between 110 and 171 °C for strain 436. However, they showed different T g : 88.82 °C for strain 540; 114.32 °C for strain B63; and 122.70 °C for strain 436. A total of eleven spots were identified by mass spectrometry, with the spore coat protein GerQ, sporulation protein YtfJ (GerW), and peptidyl-prolyl cis-trans isomerase being found in at least two strains. Altogether, the results suggested that the high survival variability of B. cereus spores to the spray drying process seems to be mainly associated with different T g and protein content. The study highlights the importance of quantifying the effects of this unit operation over the target microorganisms. These data may be relevant for the development of effective measures aiming to control the occurrence of B. cereus in milk powder as well as to reduce spoilage or safety issues associated with the presence of this bacterium in foods, particularly those formulated with milk powder., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The low survival of insect-pathogenic fungi when used for insect control in agriculture is mainly due to the deleterious effects of ultraviolet radiation and heat from solar irradiation. In this study, conidia of 15 species of entomopathogenic fungi were exposed to simulated full-spectrum solar radiation emitted by a Xenon Test Chamber Q-SUN XE-3-HC 340S (Q-LAB ® Corporation, Westlake, OH, USA), which very closely simulates full-spectrum solar radiation. A dendrogram obtained from cluster analyses, based on lethal time 50 % and 90 % calculated by Probit analyses, separated the fungi into three clusters: cluster 3 contains species with highest tolerance to simulated full-spectrum solar radiation, included Metarhizium acridum, Cladosporium herbarum, and Trichothecium roseum with LT 50  > 200 min irradiation. Cluster 2 contains eight species with moderate UV tolerance: Aschersonia aleyrodis, Isaria fumosorosea, Mariannaea pruinosa, Metarhizium anisopliae, Metarhizium brunneum, Metarhizium robertsii, Simplicillium lanosoniveum, and Torrubiella homopterorum with LT 50 between 120 and 150 min irradiation. The four species in cluster 1 had the lowest UV tolerance: Lecanicillium aphanocladii, Beauveria bassiana, Tolypocladium cylindrosporum, and Tolypocladium inflatum with LT 50  < 120 min irradiation. The QSUN Xenon Test Chamber XE3 is often used by the pharmaceutical and automotive industry to test light stability and weathering, respectively, but it was never used to evaluate fungal tolerance to full-spectrum solar radiation before. We conclude that the equipment provided an excellent tool for testing realistic tolerances of fungi to full-spectrum solar radiation of microbial agents for insect biological control in agriculture., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Survival of entomopathogenic fungi under solar ultraviolet (UV) radiation is paramount to the success of biological control of insect pests and disease vectors. The mutagenic compound 4-nitroquinoline 1-oxide (4-NQO) is often used to mimic the biological effects of UV radiation on organisms. Therefore, we asked whether tolerance to 4-NQO could predict tolerance to UV radiation in thirty isolates of entomopathogenic fungi and one isolate of a xerophilic fungus. A dendrogram obtained from cluster analyses based on the 50 and 90 % inhibitory concentrations (IC 50 and IC 90 , respectively) divided the fungal isolates into six clusters numbered consecutively based on their tolerance to 4-NQO. Cluster 6 contained species with highest tolerance to 4-NQO (IC 50  > 4.7 μM), including Mariannaea pruinosa, Lecanicillium aphanocladii, and Torrubiella homopterorum. Cluster 1 contained species least tolerant to 4-NQO (IC 50  < 0.2 μM), such as Metarhizium acridum (ARSEF 324), Tolypocladium geodes, and Metarhizium brunneum (ARSEF 7711). With few exceptions, the majority of Metarhizium species showed moderate to low tolerances (IC 50 between 0.4 and 0.9 μM) and were placed in cluster 2. Cluster 3 included species with moderate tolerance (IC 50 between 1.0 and 1.2 μM). In cluster 4 were species with moderate to high tolerance (IC 50 between 1.3 and 1.6 μM). Cluster 5 contained the species with high tolerance (IC 50 between 1.9 and 4.0 μM). The most UV tolerant isolate of M. acridum, ARSEF 324, was the least tolerant to 4-NQO. Also, L. aphanocladii, which is very susceptible to UV radiation, showed high tolerance to 4-NQO. Our results indicate that tolerance to 4-NQO does not correlate with tolerance to UV radiation. Therefore this chemical compound is not a predictor of UV tolerance in entomopathogenic fungi., (Copyright © 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Metarhizium acridum is an entomopathogen currently used against acridids. We have previously reported that exposing mycelium to visible light increases M. acridum tolerance to ultraviolet-B (UV-B) radiation. Here we evaluated if light could also increase tolerance to ultraviolet-C (UV-C) radiation. We observed that, as opposed to UV-B radiation, light did not increase tolerance to UV-C radiation under dark repair conditions. However, light did increase tolerance to UV-C radiation if photoreactivating light was present after UV-C exposure. Quantitative PCR experiments revealed that light up-regulates a photolyase gene. This is the first report showing that light regulates photoreactivating ability in M. acridum., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

On November 5th, 2015, Samarco's iron mine dam - called Fundão - spilled 50-60 million m 3 of mud into Gualaxo do Norte, a river that belongs to Rio Doce Basin. Approximately 15 km 2 were flooded along the rivers Gualaxo do Norte, Carmo and Doce, reaching the Atlantic Ocean on November 22nd, 2015. Six days after, our group collected mud, soil and water samples in Bento Rodrigues (Minas Gerais, Brazil), which was the first impacted area. Overall, the results, water samples - potable and surface water from river - presented chemical elements concentration according to Brazilian environmental legislations, except silver concentration in surface water that ranged from 1.5 to 1087 μg L -1 . In addition, water mud-containing presented Fe and Mn concentrations approximately 4-fold higher than the maximum limit for water bodies quality assessment, according to Brazilian laws. Mud particle size ranged from 1 to 200 μm. SEM-EDS spot provided us some semi quantitative data. Leaching/extraction tests suggested that Ba, Pb, As, Sr, Fe, Mn and Al have high potential mobilization from mud to water. Low microbial diversity in mud samples compared to background soil samples. Toxicological bioassays (HepG2 and Allium cepa) indicated potential risks of cytotoxicity and DNA damage in mud and soil samples used in both assays. The present study provides preliminary information aiming to collaborate to the development of future works for monitoring and risk assessment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)