Insight into the efficiency of microalgae' lipidic extracts as photosensitizers for Antimicrobial Photodynamic Therapy against Staphylococcus aureus.

Antibacterial resistance causes around 1.27 million deaths annually around the globe and has been recognized as a top 3 priority health threat. Antimicrobial photodynamic therapy (aPDT) is considered a promising alternative to conventional antibiotic treatments. Algal lipid extracts have shown antibacterial effects when used as photosensitizers (PSs) in aPDT. In this work we assessed the photodynamic efficiency of lipidic extracts of microalgae belonging to different phyla (Bacillariophyta, Chlorophyta, Cyanobacteria, Haptophyta, Ochrophyta and Rhodophyta). All the extracts (at 1 mg mL-1) demonstrated a reduction of Staphylococcus aureus >3 log10 (CFU mL-1), exhibiting bactericidal activity. Bacillariophyta and Haptophyta extracts were the top-performing phyla against S. aureus, achieving a reduction >6 log10 (CFU mL-1) with light doses of 60 J cm-2 (Bacillariophyta) and 90 J cm-2 (Haptophyta). The photodynamic properties of the Bacillariophyta Phaeodactylum tricornutum and the Haptophyta Tisochrysis lutea, the best effective microalgae lipid extracts, were also assessed at lower concentrations (75 µg mL-1, 7.5 µg mL-1, and 3.75 µg mL-1), reaching, in general, inactivation rates higher than those obtained with the widely used PSs, such as Methylene Blue and Chlorine e6, at lower concentration and light dose. The presence of chlorophyll c, which can absorb a greater amount of energy than chlorophylls a and b; rich content of polyunsaturated fatty acids (PUFAs) and fucoxanthin, which can also produce ROS, e.g. singlet oxygen (1O2), when photo-energized; a lack of photoprotective carotenoids such as ß-carotene, and low content of tocopherol, were associated with the algal extracts with higher antimicrobial activity against S. aureus. The bactericidal activity exhibited by the extracts seems to result from the photooxidation of microalgae PUFAs by the 1O2 and/or other ROS produced by irradiated chlorophylls/carotenoids, which eventually led to bacterial lipid peroxidation and cell death, but further studies are needed to confirm this hypothesis. These results revealed the potential of an unexplored source of natural photosensitizers (microalgae lipid extracts) that can be used as PSs in aPDT as an alternative to conventional antibiotic treatments, and even to conventional PSs, to combat antibacterial resistance.

Efficient Strategies to Use ß-Cationic Porphyrin-Imidazolium Derivatives in the Photoinactivation of Methicillin-Resistant Staphylococcus aureus.

Bacterial resistance to antibiotics is a critical global health issue and the development of alternatives to conventional antibiotics is of the upmost relevance. Antimicrobial photodynamic therapy (aPDT) is considered a promising and innovative approach for the photoinactivation of microorganisms, particularly in cases where traditional antibiotics may be less effective due to resistance or other limitations. In this study, two ß-modified monocharged porphyrin-imidazolium derivatives were efficiently incorporated into polyvinylpyrrolidone (PVP) formulations and supported into graphitic carbon nitride materials. Both porphyrin-imidazolium derivatives displayed remarkable photostability and the ability to generate cytotoxic singlet oxygen. These properties, which have an important impact on achieving an efficient photodynamic effect, were not compromised after incorporation/immobilization. The prepared PVP-porphyrin formulations and the graphitic carbon nitride-based materials displayed excellent performance as photosensitizers to photoinactivate methicillin-resistant Staphylococcus aureus (MRSA) (99.9999% of bacteria) throughout the antimicrobial photodynamic therapy. In each matrix, the most rapid action against S. aureus was observed when using PS 2. The PVP-2 formulation needed 10 min of exposure to white light at 5.0 µm, while the graphitic carbon nitride hybrid GCNM-2 required 20 min at 25.0 µm to achieve a similar level of response. These findings suggest the potential of graphitic carbon nitride-porphyrinic hybrids to be used in the environmental or clinical fields, avoiding the use of organic solvents, and might allow for their recovery after treatment, improving their applicability for bacteria photoinactivation.

Sulfonamide Porphyrins as Potent Photosensitizers against Multidrug-Resistant Staphylococcus aureus (MRSA): The Role of Co-Adjuvants.

Sulfonamides are a conventional class of antibiotics that are well-suited to combat infections. However, their overuse leads to antimicrobial resistance. Porphyrins and analogs have demonstrated excellent photosensitizing properties and have been used as antimicrobial agents to photoinactivate microorganisms, including multiresistant Staphylococcus aureus (MRSA) strains. It is well recognized that the combination of different therapeutic agents might improve the biological outcome. In this present work, a novel meso-arylporphyrin and its Zn(II) complex functionalized with sulfonamide groups were synthesized and characterized and the antibacterial activity towards MRSA with and without the presence of the adjuvant KI was evaluated. For comparison, the studies were also extended to the corresponding sulfonated porphyrin TPP(SO3H)4. Photodynamic studies revealed that all porphyrin derivatives were effective in photoinactivating MRSA (>99.9% of reduction) at a concentration of 5.0 µM upon white light radiation with an irradiance of 25 mW cm-2 and a total light dose of 15 J cm-2. The combination of the porphyrin photosensitizers with the co-adjuvant KI during the photodynamic treatment proved to be very promising allowing a significant reduction in the treatment time and photosensitizer concentration by six times and at least five times, respectively. The combined effect observed for TPP(SO2NHEt)4 and ZnTPP(SO2NHEt)4 with KI seems to be due to the formation of reactive iodine radicals. In the photodynamic studies with TPP(SO3H)4 plus KI, the cooperative action was mainly due to the formation of free iodine (I2).

Porphyrin Photosensitizers Grafted in Cellulose Supports: A Review.

Cellulose is the most abundant natural biopolymer and owing to its compatibility with biological tissues, it is considered a versatile starting material for developing new and sustainable materials from renewable resources. With the advent of drug-resistance among pathogenic microorganisms, recent strategies have focused on the development of novel treatment options and alternative antimicrobial therapies, such as antimicrobial photodynamic therapy (aPDT). This approach encompasses the combination of photoactive dyes and harmless visible light, in the presence of dioxygen, to produce reactive oxygen species that can selectively kill microorganisms. Photosensitizers for aPDT can be adsorbed, entrapped, or linked to cellulose-like supports, providing an increase in the surface area, with improved mechanical strength, barrier, and antimicrobial properties, paving the way to new applications, such as wound disinfection, sterilization of medical materials and surfaces in different contexts (industrial, household and hospital), or prevention of microbial contamination in packaged food. This review will report the development of porphyrinic photosensitizers supported on cellulose/cellulose derivative materials to achieve effective photoinactivation. A brief overview of the efficiency of cellulose based photoactive dyes for cancer, using photodynamic therapy (PDT), will be also discussed. Particular attention will be devoted to the synthetic routes behind the preparation of the photosensitizer-cellulose functional materials.

Evaluation of UV-C Radiation Efficiency in the Decontamination of Inanimate Surfaces and Personal Protective Equipment Contaminated with Phage ϕ6.

To help halt the global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), appropriate disinfection techniques are required. Over the last years, the interest in Ultraviolet-C (UV-C) radiation as a method to disinfect inanimate surfaces and personal protective equipment (PPE) has increased, mainly to efficiently disinfect and prevent SARS-CoV-2 from spreading and allow for the safe reuse of said equipment. The bacteriophage ϕ6 (or simply phage ϕ6) is an RNA virus with a phospholipid envelope and is commonly used in environmental studies as a surrogate for human RNA-enveloped viruses, including SARS-CoV-2. The present study investigated the use of two new UV irradiation systems ((2)2.4W and (8)5.5W)) constituted by conventional mercury UV-C lamps with a strong emission peak at ~254 nm to potentially inactivate phage ϕ6 on different surfaces (glass, plastic, stainless steel, and wood) and personal protective equipment, PPE, (surgical and filtering facepiece 2, FFP2, masks, a clear acetate visor, and disposable protective clothing). The results showed that both UV-C systems were effective in inactivating phage ϕ6, but the UV-C sterilizing chamber (8)5.5W had the best disinfection performance on the tested surfaces. The inactivation effectiveness is material-dependent on all surfaces, reaching the detection limit of the method at different times (between 60 and 240 s of irradiation). The glass surface needed less time to reduce the virus (30 s) when compared with plastic, stainless, and wood surfaces (60 s). The virus inactivation was more effective in the disposable surgical and FFP2 masks (60 and 120 s, respectively) than in the disposable vest and clear acetate visor (240 s). Overall, this study suggests that UV-C lamps with peak emission at ~254 nm could provide rapid, efficient, and sustainable sanitization procedures to different materials and surfaces. However, dosage and irradiation time are important parameters to be considered during their implementation as a tool in the fight against human coronaviruses, namely against SARS-CoV-2.

Comprehensive quality assurance for intracranial radiosurgery treatments using a postal system / Garantía de calidad para tratamientos de radiocirugía intracranial usando un sistema postal

Radiosurgery is a high-precision technique for delivering, in most cases, a single highly conformal dose to a stereotactically localized target. It can be indicated for small intracranial injury treatment, using either multiple sources of 60Co (γ rays) or high energy photon beams produced by linear accelerators. In order to minimize the impact of inaccurate localization of the target or dose delivery, a rigorous Quality Assurance (QA) program must be enforced, which should include an independent auditing system. This work proposes a simple and reliable postal QA phantom to be used as an independent evaluation. In it two important parameters were verified such as, the dosimetric precision of the planning system, by comparing the absorbed doses measured in the target volume using different dosimeters (ionization chamber, films, thermoluminescent dosimeters and L-alanine dosimeters) all calibrated against a small volume ion chamber. The exact positioning of the target volume was localized using air spaces and small steel spheres to find the appropriate target coordinates. The head phantom and the instruction sheets were extensively tested and sent by mail to selected institutions. The overall results were very encouraging and suggest that the proposed phantom may be used as a postal system as part of an independent QA tool in radiosurgery.

La radiocirugía es una técnica de alta precisión para administrar, en la mayoría de los casos, una sola dosis altamente conformada en un objetivo localizado estereotípicamente. Puede estar indicado para el tratamiento de pequeñas lesiones intracraneales, utilizando múltiples fuentes de 60Co (rayos γ) o haces de fotones de alta energía producidos por aceleradores lineales. Con el fin de minimizar el impacto de la ubicación inexacta de la administración de la meta o de la dosis, se debe aplicar un riguroso programa de control de calidad (QA), que debe incluir un sistema de auditoría independiente. Este documento propone un fantoma postal de control de calidad simple y fiable que se utilizará como evaluación independiente. Se verificó dos parámetros importantes, como la precisión dosimétrica del sistema de planificación, comparando las dosis absorbidas medidas en el volumen objetivo mediante diferentes dosis (cámara de ionización, películas, dosímetros Termoluminiscentes y dosímetros de L-alanina) todos calibrados con una pequeña cámara de iones de volumen. El posicionamiento exacto del volumen objetivo se localizó utilizando espacios aéreos y pequeñas esferas de acero para encontrar las coordenadas de destino adecuadas. El fantoma principal y las hojas de instrucciones fueron ampliamente probados y enviados por correo a instituciones seleccionadas. Los resultados generales fueron muy alentadores y sugieren que el fantoma propuesto puede utilizarse como sistema postal como parte de una herramienta independiente de control de calidad en radiocirugía.

Characterization of a Lytic Bacteriophage against Pseudomonas syringae pv. actinidiae and Its Endolysin.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen that causes canker in kiwifruit. Few conventional control methods are effective against this bacterium. Therefore, alternative approaches, such as phage therapy are warranted. In this study, a lytic bacteriophage (PN09) of Psa was isolated from surface water collected from a river in Hangzhou, China in 2019. Morphologically, PN09 was classified into the Myoviridae family, and could lyse all 29 Psa biovar 3 strains. The optimal temperature and pH ranges for PN09 activity were determined as 25 to 35 ∘C and 6.0 to 9.0, respectively. The complete genome of PN09 was found to be composed of a linear 99,229 bp double-stranded DNA genome with a GC content of 48.16%. The PN09 endolysin (LysPN09) was expressed in vitro and characterized. LysPN09 was predicted to belong to the Muraidase superfamily domain and showed lytic activity against the outer-membrane-permeabilized Psa strains. The lytic activity of LysPN09 was optimal over temperature and pH ranges of 25 to 40 ∘C and 6.0 to 8.0, respectively. When recombinant endolysin LysPN09 was combined with EDTA, Psa strains were effectively damaged. All these characteristics demonstrate that the phage PN09 and its endolysin, LysPN09, are potential candidates for biocontrol of Psa in the kiwifruit industry.

Kiwifruit bacterial canker: an integrative view focused on biocontrol strategies.

MAIN CONCLUSION: Phage-based biocontrol strategies can be an effective alternative to control Psa-induced bacterial canker of kiwifruit. The global production of kiwifruit has been seriously affected by Pseudomonas syringae pv. actinidiae (Psa) over the last decade. Psa damages both Actinidia chinensis var. deliciosa (green kiwifruit) but specially the susceptible Actinidia chinensis var. chinensis (gold kiwifruit), resulting in severe economic losses. Treatments for Psa infections currently available are scarce, involving frequent spraying of the kiwifruit plant orchards with copper products. However, copper products should be avoided since they are highly toxic and lead to the development of bacterial resistance to this metal. Antibiotics are also used in some countries, but bacterial resistance to antibiotics is a serious worldwide problem. Therefore, it is essential to develop new approaches for sustainable agriculture production, avoiding the emergence of resistant Psa bacterial strains. Attempts to develop and establish highly accurate approaches to combat and prevent the occurrence of bacterial canker in kiwifruit plants are currently under study, using specific viruses of bacteria (bacteriophages, or phages) to eliminate the Psa. This review discusses the characteristics of Psa-induced kiwifruit canker, Psa transmission pathways, prevention and control, phage-based biocontrol strategies as a new approach to control Psa in kiwifruit orchards and its advantages over other therapies, together with potential ways to bypass phage inactivation by abiotic factors.

An Insight into the Role of Non-Porphyrinoid Photosensitizers for Skin Wound Healing.

The concept behind photodynamic therapy (PDT) is being successfully applied in different biomedical contexts such as cancer diseases, inactivation of microorganisms and, more recently, to improve wound healing and tissue regeneration. The effectiveness of PDT in skin treatments is associated with the role of reactive oxygen species (ROS) produced by a photosensitizer (PS), which acts as a "double agent". The release of ROS must be high enough to prevent microbial growth and, simultaneously, to accelerate the immune system response by recruiting important regenerative agents to the wound site. The growing interest in this subject is reflected by the increasing number of studies concerning the optimization of relevant experimental parameters for wound healing via PDT, namely, light features, the structure and concentration of the PS, and the wound type and location. Considering the importance of developing PSs with suitable features for this emergent topic concerning skin wound healing, in this review, a special focus on the achievements attained for each PS class, namely, of the non-porphyrinoid type, is given.

Candida Species (Volatile) Metabotyping through Advanced Comprehensive Two-Dimensional Gas Chromatography.

Microbial metabolomics is a challenge strategy that allows a comprehensive analysis of metabolites within a microorganism and may support a new approach in microbial research, including the microbial diagnosis. Thus, the aim of this research was to in-depth explore a metabolomics strategy based on the use of an advanced multidimensional gas chromatography for the comprehensive mapping of cellular metabolites of C. albicans and non-C. albicans (C. glabrata and C. tropicalis) and therefore contributing for the development of a comprehensive platform for fungal detection management and for species distinction in early growth times (6 h). The volatile fraction comprises 126 putatively identified metabolites distributed over several chemical families: acids, alcohols, aldehydes, hydrocarbons, esters, ketones, monoterpenic and sesquiterpenic compounds, norisoprenoids, phenols and sulphur compounds. These metabolites may be related with different metabolic pathways, such as amino acid metabolism and biosynthesis, fatty acids metabolism, aromatic compounds degradation, mono and sesquiterpenoid synthesis and carotenoid cleavage. These results represent an enlargement of ca. 70% of metabolites not previously reported for C. albicans, 91% for C. glabrata and 90% for C. tropicalis. This study represents the most detailed study about Candida species exometabolome, allowing a metabolomic signature of each species, which signifies an improvement towards the construction of a Candida metabolomics platform whose application in clinical diagnostics can be crucial to guide therapeutic interventions.

Enlarging Knowledge on Lager Beer Volatile Metabolites Using Multidimensional Gas Chromatography.

Foodomics, emergent field of metabolomics, has been applied to study food system processes, and it may be useful to understand sensorial food properties, among others, through foods metabolites profiling. Thus, as beer volatile components represent the major contributors for beer overall and peculiar aroma properties, this work intends to perform an in-depth profiling of lager beer volatile metabolites and to generate new data that may contribute for molecules' identification, by using multidimensional gas chromatography. A set of lager beers were used as case-study, and 329 volatile metabolites were determined, distributed over 8 chemical families: acids, alcohols, esters, monoterpenic compounds, norisoprenoids, sesquiterpenic compounds, sulfur compounds, and volatile phenols. From these, 96 compounds are reported for the first time in the lager beer volatile composition. Around half of them were common to all beers under study. Clustering analysis allowed a beer typing according to production system: macro- and microbrewer beers. Monoterpenic and sesquiterpenic compounds were the chemical families that showed wide range of chemical structures, which may contribute for the samples' peculiar aroma characteristics. In summary, as far as we know, this study presents the most in-depth lager beer volatile composition, which may be further used in several approaches, namely, in beer quality control, monitoring brewing steps, raw materials composition, among others.

Antimicrobial Photodynamic Therapy in the Control of Pseudomonas syringae pv. actinidiae Transmission by Kiwifruit Pollen.

Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen responsible for bacterial canker in kiwifruit plants and can be disseminated through pollen. This study aimed to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) in the inactivation of Psa on kiwifruit pollen using New Methylene Blue (NMB) and Methylene Blue (MB) in the presence/absence of potassium iodide (KI). Pollen germination assays were also performed to evaluate if it was affected by aPDT. Higher reduction of Psa was achieved using NMB (5.0 µM) combined with KI (100 mM) in vitro (ca. 8 log CFU mL-1 after 90 min of irradiation), while NMB alone promoted a lower reduction (3.7 log CFU mL-1). The most efficient NMB concentration with KI was used to study the photodynamic efficiency of MB (5.0 µM). MB with KI photo-inactivated Psa more efficiently than NMB, causing the same bacterial reduction (ca. 8 log CFU mL-1) in half the irradiation time (45 min). Therefore, MB was selected for the subsequent ex vivo aPDT assays in pollen. Almost all the Psa cells added artificially to the pollen (3.2 log CFU mL-1) were photo-inactivated (3.1 log CFU mL-1), whereas aPDT had a low effect on pollen natural microorganisms. When KI was added, a significant increase in aPDT effectiveness was observed (4.5 log CFU mL-1). No negative effects were observed in the pollen germination after aPDT. The results show aPDT is an effective and safe method to Psa inactivation on kiwifruit pollen, and MB use is a promising alternative in the control of Psa transmission.

Combined Application of Bacteriophages and Carvacrol in the Control of Pseudomonas syringae pv. actinidiae Planktonic and Biofilm Forms.

Pseudomonas syringae pv. actinidiae (Psa) is the causative agent of the bacterial canker of kiwifruit (Actinidia spp.). Phage therapy has been suggested as a viable alternative approach to controlling this disease, but its efficacy is limited by the emergence of phage-resistant mutants. Carvacrol is an essential oil that may be useful for the control of Psa. Combination therapies can be used to overcome resistance development. Here, the combination of phages (single phage suspensions of phages PN05 and PN09, and a cocktail of both phages) and carvacrol was investigated in controlling Psa planktonic and biofilm forms in vitro. The phage therapy alone (with phages PN05 and PN09), and the carvacrol alone (minimum inhibitory concentration 2.0 mg/mL), inhibited Psa growth, but the combined effect of both therapies was more effective. The phages alone effectively inhibited Psa growth for 24 h, but Psa regrowth was observed after this time. The carvacrol (2.0 mg/mL) alone prevented the biofilm formation for 48 h, but did not destroy the pre-formed biofilms. The combined treatment, phages and carvacrol (2.0 mg/mL), showed a higher efficacy, preventing Psa regrowth for more than 40 h. In conclusion, the combined treatment with phages and carvacrol may be a promising, environment-friendly and cost-effective approach to controlling Psa in the kiwifruit industry.

Unsymmetrical cationic porphyrin-cyclodextrin bioconjugates for photoinactivation of Escherichia coli.

Photodynamic inactivation (PDI) of microorganisms has been used for the treatment of bacterial infection. PDI is based on the combination of three non-toxic elements: a photosensitizer (PS), light and molecular oxygen, which lead to the formation of reactive oxygen species (ROS) that cause lethal oxidative damage into the target pathogenic bacteria. For that, clinical approved tetrapyrrolic macrocycles, with particular emphasis on photoactive porphyrin (Por) dyes, have been used as PS in PDI for different biomedical applications. Two novel unsymmetrical free-base thiopyridyl Pors conjugated with α- or γ-CD units (Pors 2 and 3) were prepared and the corresponding cationic ones (Pors 2a and 3a) were assessed as water-soluble photosensitizer (PS) agents by photophysical, photochemical and E. coli photobiological studies. The presence of the CD unit and the positive charges on the Por periphery (2a and 3a) enhance their solubility in aqueous media. The photoactivity of the two cationic Pors 2a and 3a ensures their potential as PDI drugs against Gram-negative bacteria model, a bioluminescent E. coli, which the best PDI efficiency was determined for Por 3a that achieved the highest bacterial reduction of 4.0 log10 (ANOVA, p < 0.0001), reaching the detection limit of the method after 15 min.

Use of phage ϕ6 to inactivate Pseudomonas syringae pv. actinidiae in kiwifruit plants: in vitro and ex vivo experiments.

Over the last years, the global production and trade of kiwifruit has been severely impacted by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen that causes a disease in kiwifruit plants known as bacterial canker. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with disinfectants, copper-based bactericides and/or antibiotics. Moreover, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Phage therapy may be an alternative approach to inactivate Psa. The present study investigated the potential application of the already commercially available bacteriophage (or phage) ϕ6 to control Psa infections. The inactivation of Psa was assessed in vitro, using liquid culture medium, and ex vivo, using artificially contaminated kiwifruit leaves with two biovar 3 (a highly aggressive pathogen) strains (Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10). In the in vitro experiments, the phage ϕ6 was effective against both strains (maximum reduction of 2.2 and 1.9 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). In the ex vivo tests, the decrease was lower (maximum reduction 1.1 log and 1.8 CFU/mL for Psa CRA-FRU 12.54 and Psa CRA-FRU 14.10, respectively). The results of this study suggest that the commercially available phage ϕ6 can be an effective alternative to control Psa infections in kiwifruit orchards.

Efficiency of Phage φ6 for Biocontrol of Pseudomonas syringae pv. syringae: An in Vitro Preliminary Study.

Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees. The most common treatments for biocontrol of P. syringae pv. syringae infections has involved copper derivatives and/or antibiotics. However, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Therefore, it is essential to search for new approaches for controlling P. syringae pv. syringae. Phage therapy can be a useful alternative tool to the conventional treatments to control P. syringae pv. syringae infections in plants. In the present study, the efficacy of bacteriophage (or phage) φ6 (a commercially available phage) was evaluated in the control of P. syringae pv. syringae. As the plants are exposed to the natural variability of physical and chemical parameters, the influence of pH, temperature, solar radiation and UV-B irradiation on phage φ6 viability was also evaluated in order to develop an effective phage therapy protocol. The host range analysis revealed that the phage, besides its host (P. syringae pv. syringae), also infects the Pseudomonas syringae pv. actinidiae CRA-FRU 12.54 and P. syringae pv. actinidiae CRA-FRU 14.10 strains, not infecting strains from the other tested species. Both multiplicities of infection (MOIs) tested, 1 and 100, were effective to inactivate the bacterium, but the MOI 1 (maximum reduction of 3.9 log CFU/mL) was more effective than MOI 100 (maximum reduction of 2.6 log CFU/mL). The viability of phage φ6 was mostly affected by exposure to UV-B irradiation (decrease of 7.3 log PFU/mL after 8 h), exposure to solar radiation (maximum reduction of 2.1 PFU/mL after 6 h), and high temperatures (decrease of 8.5 PFU/mL after 6 days at 37 °C, but a decrease of only 2.0 log PFU/mL after 67 days at 15 °C and 25 °C). The host range, high bacterial control and low rates of development of phage-resistant bacterial clones (1.20 × 10-3) suggest that this phage can be used to control P. syringae pv. syringae infections in plants, but also to control infections by P. syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit. Although the stability of phage φ6 was affected by UV-B and solar radiation, this can be overcome by the application of phage suspensions at the end of the day or at night.

Photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit plants by cationic porphyrins.

MAIN CONCLUSION: The studied cationic porphyrins formulation allows an effective photoinactivation of Pseudomonas syringae pv. actinidiae in kiwifruit leaves under sunlight irradiation, without damaging the plant. Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative phytopathogenic bacterium responsible for canker on kiwifruit plant. Over the last decade, this bacterium dramatically affected the production of this fruit worldwide, causing significant economic losses. In general, Psa control consists in the application of copper which are toxic and persist in the environment. The application of antimicrobial photodynamic therapy (aPDT) as an alternative to inactivate Psa has already been demonstrated in recent studies that showed a 4 log Psa reduction using the cationic porphyrin Tetra-Py+-Me as photosensitizer (PS) and 3 consecutive cycles of treatment with a light irradiance of 150 mW cm-2. The present work aimed to evaluate the photodynamic efficiency of a new formulation constituted with five cationic porphyrins as PS in Psa inactivation. This new formulation was prepared to have as main component the tri-cationic porphyrin which is considered one of the most efficient photosensitizers in the photoinactivation of microorganisms. The in vitro study with a PS concentration of 5.0 µM and low irradiance, showed a 7.4 log photoinactivation after 60 min. Posteriorly, several assays were performed with the PS at 50 µM on kiwifruit leaves (ex vivo), under different conditions of light and inoculation. The ex vivo assays with artificially contaminated leaves showed a 2.8 and 4.5 log inactivation with low irradiance and sunlight, respectively, after 90 min. After a second treatment with sunlight, a 6.2 log inactivation was achieved. The photoinactivation on naturally contaminated leaves was about 2.3 log after 90 min sunlight irradiation. Ten consecutive cycles of phototreatment in sub-lethal conditions showed that Psa does not develop resistance, nor recover viability. The results suggest that aPDT can be an alternative to the current methods used to control Psa, since it was possible to inactivate this bacterium under sunlight, without damaging the leaves.

An insight into the photodynamic approach versus copper formulations in the control of Pseudomonas syringae pv. actinidiae in kiwi plants.

In the last decade, the worldwide production of kiwi fruit has been highly affected by Pseudomonas syringae pv. actinidiae (Psa), a phytopathogenic bacterium; this has led to severe economic losses that are seriously affecting the kiwi fruit trade. The available treatments for this disease are still scarce, with the most common involving frequently spraying the orchards with copper derivatives, in particular cuprous oxide (Cu2O). However, these copper formulations should be avoided due to their high toxicity; therefore, it is essential to search for new approaches for controlling Psa. Antimicrobial photodynamic therapy (aPDT) may be an alternative approach to inactivate Psa. aPDT consists in the use of a photosensitizer molecule (PS) that absorbs light and by transference of the excess of energy or electrons to molecular oxygen forms highly reactive oxygen species (ROS) that can affect different molecular targets, thus being very unlikely to lead to the development of microbe resistance. The aim of the present study was to evaluate the effectiveness of aPDT to photoinactivate Psa, using the porphyrin Tetra-Py+-Me and different light intensities. The degree of inactivation of Psa was assessed using the PS at 5.0 µM under low irradiance (4.0 mW cm-2). Afterward, ex vivo experiments, using artificially contaminated kiwi leaves, were conducted with a PS at 50 µM under 150 mW cm-2 and sunlight irradiation. A reduction of 6 log in the in vitro assays after 90 min of irradiation was observed. In the ex vivo tests, the decrease was lower, approximately 1.8 log reduction at an irradiance of 150 mW cm-2, 1.2 log at 4.0 mW cm-2, and 1.5 log under solar radiation. However, after three successive cycles of treatment under 150 mW cm-2, a 4 log inactivation was achieved. No negative effects were observed on leaves after treatment. Assays using Cu2O were also performed at the recommended concentration by law (50 g h L-1) and at concentrations 10 times lower, in which at both concentrations, Psa was efficiently inactivated (5 log inactivation) after a few minutes of treatment, but negative effects were observed on the leaves after treatment.

Metabolomics strategy for the mapping of volatile exometabolome from Saccharomyces spp. widely used in the food industry based on comprehensive two-dimensional gas chromatography.

Saccharomyces spp. are widely used in the food and beverages industries. Their cellular excreted metabolites are important for general quality of products and can contribute to product differentiation. This exploratory study presents a metabolomics strategy for the comprehensive mapping of cellular metabolites of two yeast species, Saccharomyces cerevisiae and S. pastorianus (both collected in an industrial context) through a multidimensional chromatography platform. Solid-phase microextraction was used as a sample preparation method. The yeast viability, a specific technological quality parameter, was also assessed. This untargeted analysis allowed the putative identification of 525 analytes, distributed over 14 chemical families, the origin of which may be explained through the pathways network associated with yeasts metabolism. The expression of the different metabolic pathways was similar for both species, event that seems to be yeast genus dependent. Nevertheless, these species showed different growth rates, which led to statistically different metabolites content. This was the first in-depth approach that characterizes the headspace content of S. cerevisiae and S. pastorianus species cultures. The combination of a sample preparation method capable of providing released volatile metabolites directly from yeast culture headspace with comprehensive two-dimensional gas chromatography was successful in uncovering a specific metabolomic pattern for each species.

An effective and potentially safe blood disinfection protocol using tetrapyrrolic photosensitizers.

AIM: Conventional disinfection techniques, considered safe for plasma, are usually associated with collateral damages on concentrated platelets and erythrocytes. Alternative methods are required and antimicrobial photodynamic therapy (aPDT) seems promising. In this study the effectiveness of two photosensitizers (PS), a porphyrin and a phthalocyanine, to disinfect blood products was evaluated. RESULTS: The cationic porphyrin was more effective in the photoinactivation of bacteria. Also, no significant osmotic stress was found for samples treated with PS at 5.0 µM in isotonic conditions after antimicrobial photodynamic therapy. CONCLUSION: Effective reduction of Gram-positive bacteria at 5.0 µM of PS provided promising indications toward its safe use to disinfect blood samples. For Gram-negative bacteria, lower PS concentrations, between 5.0 and 10 µM, must be tested.