M. G. P. M. S. Neves

Porphyrin derivatives as photosensitizers for the inactivation of Bacillus cereus endospores.

Aims:  In this study, we propose (i) to study the photodynamic inactivation (PDI) efficiency of neutral and cationic porphyrin derivatives, (ii) to characterize the kinetics of the inactivation process using Bacillus cereus as a model endospore‐producing bacterium and (iii) to conclude on the applicability of porphyrin derivatives in the inactivation of bacterial endospores.

Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

Oxidation of monoterpenes with hydrogen peroxide catalysed by Keggin-type tungstoborates

The Keggin-type anions [MnIII(H2O)BW11O39]6− and [BW12O40]5− were used for the first time as catalysts in studies of oxidation of monoterpenes by hydrogen peroxide. The oxidation of geraniol 1, nerol 2, (+)-3-carene 3, thymol 4 and carvacrol 5 was examined. The reactions were carried out in acetonitrile, at room temperature (for 1 and 2) or at reflux (for 3, 4 and 5). Compounds 1 and 2 were preferentially epoxidised at the C2–C3 double bond, whereas 3, under controlled reaction conditions, could afford only the α-epoxide. The oxidation of 4 and 5 yielded a mixture of benzoquinones. For tetrabutylammonium salts, the MnIII substituted anion was found to be a more efficient catalyst than [BW12O40]5−. The infrared spectra of the recovered solids at the end of reaction showed that the Keggin anions were still present, but the electronic spectra of the manganese residues indicate that oxidation of MnIII had taken place.

Influence of external bacterial structures on the efficiency of photodynamic inactivation by a cationic porphyrin

The main targets of photodynamic inactivation (PDI) are the external bacterial structures, cytoplasmic membrane and cell wall. In this work it was evaluated how the external bacterial structures influence the PDI efficiency. To reach this objective 8 bacteria with distinct external structures were selected; 4 Gram-negative bacteria (Escherichia coli, with typical Gram-negative external structures; Aeromonas salmonicida, Aeromonas hydrophila both with an S-layer and Rhodopirellula sp., with a peptidoglycan-less proteinaceous cell wall and with cytoplasm compartmentalization) and 4 Gram-positive bacteria (Staphylococcus aureus, with typical Gram-positive external structures; Truepera radiovictrix, Deinococcus geothermalis and Deinococcus radiodurans, all with thick cell walls that give them Gram-positive stains, but including a second complex multi-layered membrane and structurally analogous to that of Gram-negative bacteria). The studies were performed in the presence of 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetraiodide (Tetra-Py(+)-Me) at 5.0 μM with white light (40 W m(-2)). The susceptibility of each bacteria to PDI by Tetra-Py(+)-Me was dependent on bacteria external structures. Although all Gram-positive bacteria were inactivated to the detection limit (reduction of ∼8 log) after 60-180 min of irradiation, the inactivation followed distinct patterns. Among the Gram-negative bacteria, E. coli was the only species to be inactivated to the detection limit (∼8 log after 180 min). The efficiency of inactivation of the two species of Aeromonas was similar (reduction of ∼5-6 log after 270 min). Rhodopirellula was less susceptible (reduction of ∼4 log after 270 min). As previously observed, the Gram-positive bacteria are more easily inactivated than Gram-negative strains, and this is even true for T. radiovictrix, D. geothermalis and D. radiodurans, which have a complex multi-layered cell wall. The results support the theory that the outer cell structures are major bacterial targets for PDI. Moreover, the chemical composition of the external structures has a stronger effect on PDI efficiency than complexity and the number of layers of the external coating, and lipids seem to be an important target of PDI.

Photoinactivation of Escherichia coli (SURE2) without intracellular uptake of the photosensitizer

This study was performed to investigate the possibility to photodynamically inactivate Gram‐negative bacteria without intracellular uptake of the photosensitizer. The efficiency of the photodynamic growth inhibition of Escherichia coli (SURE2) was proved in a comparative study of a neutral and a cationic photosensitizer.

New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.

Fluorescent Bioactive Corrole Grafted-Chitosan Films.

Transparent corrole grafted-chitosan films were prepared by chemical modification of chitosan with a corrole macrocycle, namely, 5,10,15-tris(pentafluorophenyl)corrole (TPFC), followed by solvent casting. The obtained films were characterized in terms of absorption spectra (UV-vis), FLIM (fluorescence lifetime imaging microscopy), structure (FTIR, XPS), thermal stability (TGA), thermomechanical properties (DMA), and antibacterial activity. The results showed that the chemical grafting of chitosan with corrole units did not affect its film-forming ability and that the grafting yield increased with the reaction time. The obtained transparent films presented fluorescence which increases with the amount of grafted corrole units. Additionally, all films showed bacteriostatic effect against S. aureus, as well as good thermomechanical properties and thermal stability. Considering these features, promising applications may be envisaged for these corrole-chitosan films, such as biosensors, bioimaging agents, and bioactive optical devices.

Effect of Photodynamic Therapy on the Virulence Factors of Staphylococcus aureus

Staphylococcus aureus is a Gram-positive bacterium that is present in the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus strains, new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation (PDI) process is based in the combined use of light, oxygen, and an intermediary agent (a photosensitizer). These three components interact to generate cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. Although PDI is being shown to be a promising alternative to the antibiotic approach for the inactivation of pathogenic microorganisms, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus and to assess the potential development of resistance of this bacterium as well as the recovery of the expression of the virulence factors after successive PDI cycles. For this, the photosensitizer 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus [one reference strain, one strain with one enterotoxin, two strains with three enterotoxins and two methicillin resistant strains (MRSA) – one with five enterotoxins and the other without enterotoxins] were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production, and resistance/susceptibility to methicillin was tested. To assess the development of resistance after successive cycles of treatment, three strains of S. aureus (ATCC 6538, 2065 MA, and SA 3 MRSA) were used. The surviving colonies of a first cycle of PDI were collected from the solid medium and subjected to further nine consecutive cycles of PDI. The results indicate that the expression of some external virulence factors is affected by PDI and enterotoxin producing strains were more susceptible to PDI than non-toxigenic strains. The surviving bacteria did not develop resistance. PDI, contrarily to traditional antibiotics, inhibited the expression of virulence factors, efficiently inactivating either highly virulent strains and low virulent S. aureus strains, inactivating also antibiotic susceptible and resistant strains, without development of photoresistance after at least 10 consecutive cycles of treatment, and so this therapy may become a strong promising alternative to antibiotics to control pathogenic microorganisms.

Photo-oxygenation of meso-tetraphenylporphyrin derivatives: the influence of the substitution pattern and characterization of the reaction products

The photo-oxidation reactions of several meso-tetraphenylporphyrins have been studied in order to ascertain the chemical stability of this class of compounds towards singlet oxygen. The 2,6-disubstituted ones showed an excellent stability, whilst this is not the case for other porphyrins with different substitution pattern on the phenyl rings. This parallels what has been previously found using mono-oxygenated donors. The steric effects in protecting the macrocycle seems to be predominant over the electronic ones; even with electron-donating groups in the 2,6-positions, e.g. methoxy groups, the porphyrin macrocycle was not degraded. The reaction on the β-tetrabrominated meso-tetraphenylporphyrin also proceeded easily and yielded a bilinone derivative. The structures of the final products were all elucidated by mass, 1H and 13CNMR spectroscopy.

Bioluminescence and its application in the monitoring of antimicrobial photodynamic therapy

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.