Showing papers by "Maria A. F. Faustino published in 2011"

Mechanisms of photodynamic inactivation of a Gram-negative recombinant bioluminescent bacterium by cationic porphyrins

Abstract: Photodynamic therapy is a very promising approach to inactivate pathogenic microorganisms. The photodamage of cells involves reactive oxygen species (ROS) which are generated in situ by two main mechanisms (type I and/or type II). The mechanism responsible for the photoinactivation (PI) of a bioluminescent recombinant Escherichia coli, induced by three different cationic porphyrins, was identified in this work using a rapid method based on the monitoring of the metabolic activity of this bacterium. The inhibitory effect of the photodynamic process in the presence of a singlet oxygen quencher (sodium azide) or free radical scavengers (D-mannitol and L-cysteine) was evaluated by exposing bacterial suspensions with 0.5 μM Tri-Py+-Me-PF, 5.0 μM Tetra-Py+-Me or 5.0 μM Tri-SPy+-Me-PF to white light. Strong bacterial protection was observed with sodium azide (100 mM) for the three cationic porphyrins. However, in the presence of Tri-Py+-Me-PF and Tetra-Py+-Me and the free radical scavengers (L-cysteine and D-mannitol) the reduction on the bacterial bioluminescence was significantly higher and similar to that obtained in their absence (5.4–6.0 log reduction). In the case of Tri-SPy+-Me-PF two distinct behaviours were observed when L-cysteine and D-mannitol were used as free radical scavengers: while the presence of L-cysteine (100 mM) lead to a bacterial protection similar to the one observed with sodium azide, in the presence of D-mannitol only a small protection was detected. The high inhibition of the PS activity by L-cysteine is not due to its radical scavenger ability but due to the singlet oxygen quenching by the sulfanyl group (–SH). In fact, the photodecomposition of 1,3-diphenylisobenzofuran in the presence of Tri-SPy+-Me-PF is completely suppressed when L-cysteine is present. The results obtained in this study suggest that singlet oxygen (type II mechanism) plays a very important role over free radicals (type I mechanism) on the PI process of the bioluminescent E. coli by Tri-Py+-Me-PF, Tetra-Py+-Me and Tri-SPy+-Me-PF. Although the use of scavengers is an adequate and simple approach to evaluate the relative importance of the two pathways, it is important to choose scavengers which do not interfere in both PI mechanisms. Sodium azide and D-mannitol seem to be good oxygen and free radical quenchers, respectively, to study the PI mechanisms by porphyrinic photosensitizers.

Photodynamic inactivation of Penicillium chrysogenum conidia by cationic porphyrins.

Abstract: This work reports the photophysical and biological evaluation of five cationic porphyrins as photosensitizers (PS) for the photodynamic inactivation (PDI) of Penicillium chrysogenum conidia. Two different cationic porphyrin groups were synthesized from 5,10,15,20-tetrakis(4-pyridyl)porphyrin and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin. The photostability and singlet oxygen generation studies showed that these molecules are photostable and efficient singlet oxygen generators. PDI experiments of P. chrysogenum conidia conducted with 50 μmol L−1 of photosensitiser under white light at a fluence rate of 200 mW cm−2 over 20 min showed that the most effective PS caused a 4.1 log reduction in the concentration of viable conidia. The present results show that porphyrins 1a and 1b are more efficient PSs than porphyrin 2a while porphyrins 1c and 2b show no inactivation of P. chrysogenum. It is also clear that the effectiveness of the molecule as PS for antifungal PDI is strongly related with the porphyrin substituent groups, and consequently their solubility in physiological media. The average amount of PS adsorbed per viable conidium was a determining factor in the photoinactivation efficiency and varied between the different studied PSs. Cationic PSs 1a and 1b might be promising anti-fungal PDI agents with potential applications in phytosanitation, biofilm control, bioremediation, and wastewater treatment.

Photodynamic Antimicrobial Chemotherapy in Aquaculture: Photoinactivation Studies of Vibrio fischeri

Abstract: BACKGROUND: Photodynamic antimicrobial chemotherapy (PACT) combines light, a light-absorbing molecule that initiates a photochemical or photophysical reaction, and oxygen The combined action of these three components originates reactive oxygen species that lead to microorganisms' destruction The aim was to evaluate the efficiency of PACT on Vibrio fischeri: 1) with buffer solution, varying temperature, pH, salinity and oxygen concentration values; 2) with aquaculture water, to reproduce photoinactivation (PI) conditions in situ METHODOLOGY/ PRINCIPAL FINDINGS: To monitor the PI kinetics, the bioluminescence of V fischeri was measured during the experiments A tricationic meso-substituted porphyrin (Tri-Py(+)-Me-PF) was used as photosensitizer (5 µM in the studies with buffer solution and 10-50 µM in the studies with aquaculture water); artificial white light (4 mW cm(-2)) and solar irradiation (40 mW cm(-2)) were used as light sources; and the bacterial concentration used for all experiments was ≈10(7) CFU mL(-1) (corresponding to a bioluminescence level of 10(5) relative light units--RLU) The variations in pH (65-85), temperature (10-25°C), salinity (20-40 g L(-1)) and oxygen concentration did not significantly affect the PI of V fischeri, once in all tested conditions the bioluminescent signal decreased to the detection limit of the method (≈7 log reduction) The assays using aquaculture water showed that the efficiency of the process is affected by the suspended matter Total PI of V fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of Tri-Py(+)-Me-PF CONCLUSIONS/ SIGNIFICANCE: If PACT is to be used in environmental applications, the matrix containing target microbial communities should be previously characterized in order to establish an efficient protocol having into account the photosensitizer concentration, the light source and the total light dose delivered The possibility of using solar light in PACT to treat aquaculture water makes this technology cost-effective and attractive

Applicability of photodynamic antimicrobial chemotherapy as an alternative to inactivate fish pathogenic bacteria in aquaculture systems

Abstract: Aquaculture activities are increasing worldwide, stimulated by the progressive reduction of natural fish stocks in the oceans. However, these activities also suffer heavy production and financial losses resulting from fish infections caused by microbial pathogens, including multidrug resistant bacteria. Therefore, strategies to control fish infections are urgently needed, in order to make aquaculture industry more sustainable. Antimicrobial photodynamic therapy (aPDT) has emerged as an alternative to treat diseases and prevent the development of antibiotic resistance by pathogenic bacteria. The aim of this work was to evaluate the applicability of aPDT to inactivate pathogenic fish bacteria. To reach this objective a cationic porphyrin Tri-Py+-Me-PF was tested against nine pathogenic bacteria isolated from a semi-intensive aquaculture system and against the cultivable bacteria of the aquaculture system. The ecological impact of aPDT in the aquatic environment was also tested on the natural bacterial community, using the overall bacterial community structure and the cultivable bacteria as indicators. Photodynamic inactivation of bacterial isolates and of cultivable bacteria was assessed counting the number of colonies. The impact of aPDT in the overall bacterial community structure of the aquaculture water was evaluated by denaturing gel gradient electrophoresis (DGGE). The results showed that, in the presence of Tri-Py+-Me-PF, the growth of bacterial isolates was inhibited, resulting in a decrease of ≈7–8 log after 60–270 min of irradiation. Cultivable bacteria were also considerably affected, showing decreases up to the detection limit (≈2 log decrease on cell survival), but the inactivation rate varied significantly with the sampling period. The DGGE fingerprint analyses revealed changes in the bacterial community structure caused by the combination of aPDT and light. The results indicate that aPDT can be regarded as a new approach to control fish infections in aquaculture systems, but it is clearly more difficult to inactivate the complex natural bacterial communities of aquaculture waters than pure cultures of bacteria isolated from aquaculture systems. Considering the use of aPDT to inactivate pathogenic microbial community of aquaculture systems the monitoring of microorganisms is needed in order to select the most effective conditions.

Bioluminescence and its application in the monitoring of antimicrobial photodynamic therapy.

Abstract: Light output from bioluminescent microorganisms is a highly sensitive reporter of their metabolic activity and therefore can be used to monitor in real time the effects of antimicrobials. Antimicrobial photodynamic therapy (aPDT) is receiving considerable attention for its potentialities as a new antimicrobial treatment modality. This therapy combines oxygen, a nontoxic photoactive photosensitizer, and visible light to generate reactive oxygen species (singlet oxygen and free radicals) that efficiently destroy microorganisms. To monitor this photoinactivation process, faster methods are required instead of laborious conventional plating and overnight incubation procedures. The bioluminescence method is a very interesting approach to achieve this goal. This review covers recent developments on the use of microbial bioluminescence in aPDT in the clinical and environmental areas.

Vilsmeier-Haack formylation of Cu(II) and Ni(II) porphyrin complexes under microwaves irradiation

Abstract: Microwave irradiation is a powerful tool in organic synthesis allowing for instance, to reduce reaction times and to obtain the desired products in better yields. Contrary to what is reported in literature, the first Vilsmeier-Haack formylation of Ni(II) and Cu(II) complexes of meso-tetraarylporphyrins by using microwave irradiation is described here. Different solvents, irradiation powers and reaction times were studied using the Ni(II) and Cu(II) complexes of 5,10,15,20-tetraphenylporphyrin and the best found conditions were extended to a series of other meso-substituted tetraarylporphyrins. The products, namely the new ones were isolated in good to excellent yields and with a significant reduction on the reaction times. Scaled-up studies have shown that the efficiency of the process is not affected by increasing the amount of porphyrin.

Porphyrin–Phosphoramidate Conjugates: Synthesis, Photostability and Singlet Oxygen Generation

Abstract: meso-Tetrakis(pentafluorophenyl)porphyrin reacts with aminoalkylphosphoramidates to afford porphyrins substituted with one or four phosphoramidate groups in the 4-position of the meso-aryl groups. The new porphyrin derivatives show high photostability and some are better singlet oxygen generators than meso-tetrakis(1-methylpyridinium-4-yl)porphyrin, a well known good singlet oxygen producer.

Reaction of β-Vinyl-meso-tetraphenylporphyrin with o-Quinone Methides

Abstract: The hetero-Diels-Alder reaction of β-vinyl-meso-­tetraphenylporphyrinatozinc(II) with quinone methides generated in situ from Knoevenagel reaction of 2-hydroxy-1,4-naphthoqui­none, 4-hydroxycoumarin, and 4-hydroxy-6-methylcoumarin with paraformaldehyde and directly from o-hydroxybenzyl alcohol derivatives is reported.