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.

Potential cannabidiol (CBD) repurposing as antibacterial and promising therapy of CBD plus polymyxin B (PB) against PB-resistant gram-negative bacilli.

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.

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.

Efficient photodynamic inactivation of Candida albicans by porphyrin and potassium iodide co-encapsulation in micelles.

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.

Responsiveness of entomopathogenic fungi to menadione-induced oxidative stress.

Entomopathogenic fungi are predisposed to ROS induced by heat and UV-A radiation when outside the insect host. When inside the host, they are subject to phagocytic cells that generate ROS to eliminate invading pathogens. The oxidative stress tolerance of the entomopathogenic fungi Aschersonia aleyrodis (ARSEF 430 and 10276), Aschersonia placenta (ARSEF 7637), Beauveria bassiana (ARSEF 252), Isaria fumosorosea (ARSEF 3889), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium acridum (ARSEF 324), Metarhizium anisopliae (ARSEF 5749), Metarhizium brunneum (ARSEF 1187 and ARSEF 5626), Metarhizium robertsii (ARSEF 2575), Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), and Simplicillium lanosoniveum (ARSEF 6430 and ARSEF 6651) was studied based on conidial germination on a medium supplemented with menadione. Conidial germination was evaluated 24 h after inoculation on potato dextrose agar (PDA) (control) or PDA supplemented with menadione. The two Aschersonia species (ARSEF 430, 7637, and 10276) were the most susceptible fungi, followed by the two Tolypocladium species (ARSEF 3392 and 4877) and the M. acridum (ARSEF 324). Metarhizium brunneum (ARSEF 5626) and M. anisopliae (ARSEF 5749) were the most tolerant isolates with MIC 0.28 mM. All fungal isolates, except ARSEF 5626 and ARSEF 5749, were not able to germinate at 0.20 mM.