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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.
... In the context of optimizing PSs for specific applications [24,28], a detailed characterization and comparison in vitro [31][32][33] as well as in in vivo models [34][35][36] for infections with multidrug-resistant pathogens is the subject of current research. The PSs currently used are almost exclusively from the groups of porphyrins, bacteriochlorins, chlorins, and phthalocyanins [14,37]. ...
... The PSs currently used are almost exclusively from the groups of porphyrins, bacteriochlorins, chlorins, and phthalocyanins [14,37]. A particularly pronounced efficacy against Gram-negative bacteria has been described for the cationic PSs such as TMPyP and THTPS [30,33,[38][39][40][41]. In contrast, anionic porphyrin derivatives have been reported to be effective for tumor treatment [24]. ...
... Our results confirm the previously reported antimicrobial effects of TMPyP-based photodynamic inactivation of ESBL-producing E. coli in vitro [33,40,41,56]. Overall, porphyrinoidbased PS are considered as non-toxic and well-tolerated at concentrations that are required for antimicrobial PDT [67]. ...
Porphyrinoid-based photodynamic inactivation (PDI) provides a promising approach to treating multidrug-resistant infections. However, available agents for PDI still have optimization potential with regard to effectiveness, toxicology, chemical stability, and solubility. The currently available photosensitizer TMPyP is provided with a para substitution pattern (para-TMPyP) of the pyridinium groups and has been demonstrated to be effective for PDI of multidrug-resistant bacteria. To further improve its properties, we synthetized a structural variant of TMPyP with an isomeric substitution pattern in a meta configuration (meta-TMPyP), confirmed the correct structure by crystallographic analysis and performed a characterization with NMR-, UV/Vis-, and IR spectroscopy, photostability, and singlet oxygen generation assay. Meta-TMPyP had a hypochromic shift in absorbance (4 nm) with a 55% higher extinction coefficient and slightly improved photostability (+6.9%) compared to para-TMPyP. Despite these superior molecular properties, singlet oxygen generation was increased by only 5.4%. In contrast, PDI, based on meta-TMPyP, reduced the density of extended spectrum β-lactamase-producing and fluoroquinolone-resistant Escherichia coli by several orders of magnitude, whereby a sterilizing effect was observed after 48 min of illumination, while para-TMPyP was less effective (p < 0.01). These findings demonstrate that structural modification with meta substitution increases antibacterial properties of TMPyP in PDI.
... [9][10][11] However,t he same modes of action of PDT and aPDT raise a great challenge in selectively inactivating bacterialc ells but leavingm ammalian cells unaffected.U nlike mammalian cells, in whicha cidic phospholipidsa re mainly located in the inner leafleto fp lasma membranes, acidic phospholipids andn egatively chargedc omponents, such as teichuronic acid (for Gram positive bacteria, for example, S. aureus)a nd lipopolysaccharide (for Gram negative bacteria, for example, E. coli)a re mainly locatedi nt he outer edges of cell walls or outer membranes, making the bacterial surface highly negatively charged. [12,13] This disparity has been leveraged to develop cationic aPDT agents, such as triarylmethanes [14,15] and borondipyrromethene (BODIPY)d yes bearing two positive charges, [16] peryleneb earing four positive charges, [17] porphyrins, [18][19][20][21][22][23] phthalocyanines [24] and bacteriochlorin [25,26] bearing three, four, or eight positive charges, with the aim to achieve higher binding affinity toward bacteria.T hough these photosensitizers showed remarkable inactivation capability against bacterial cells, in several cases, phototoxicity towardm ammalianc ells was observed, [14,23] suggestingl imited selectivity of the aPDT agents. For example,atriarylmethane dye reduced CFU of E. coli by 5log units at 1 mm upon irradiation, but only 60 %A 549 cells were alive undert he same conditions. ...
... Hamblin and co-workers foundc ationic porphyrins were taken up more rapidly by microbial cells than mammalianc ells, whichw as utilized to improve selectivityb yas hort incubation time (30 min), [25,26] but will complicate clinicala pplication. In most cases, [16][17][18][19][20][21][22]24] the selectivity between bacterial and mammalian cells of theseaPDT agentswas not reported. ...
... In this work, using quaternary ammonium-modified bipyridine as ligand,t hree new Ru II complexes Ru1-Ru3 with four,s ix, and eight positive charges ( Figure 1) weree asily prepared. The spherical octahedral coordination structure and hydrophilic cationicc haracter of the Ru II core may distinguish them from the reported organic aPDT agents, [14][15][16][17][18][19][20][21][22][23] which generally have large planar conjugated and highly hydrophobic skeletons, making these Ru II complexes efficiently bind to bacteria but not mammalian cells. Our results indicatet hat complex Ru3 bearing eight positive charges exhibited the most potent aPDT activity against S. aureus,i ncluding MRSA. ...
Ruthenium(II) polypyridyl complexes featuring peripheral quaternary ammonium structures were found to be able to selectively inactivate Gram‐positive Staphylococcus aureus (S. aureus), including methicillin‐resistant S. aureus (MRSA) upon visible light irradiation, but have low phototoxicity toward 293T cells, L02 cells and lack hemolysis toward rabbit red blood cells (RBC), exhibiting promising potential as a novel type of antimicrobial photodynamic therapy (aPDT) agents.
... Bioluminescent recombinant E. coli was also an important tool to identify the mechanisms of aPDT with promising antimicrobial agents developed in our group: the tricationic porphyrins Tri-Py(+)-Me (Fig. 6 ) [ 83,97,99 ] and the 5-(pentafluorophenyl)-10,15,20tris[2,3,5,6-tetrafluoro-4-(1-methylpyridinium-4-ylsulfanyl)phenyl]porphyrin tri-iodide (Tri-SPy+-Me-PF) [ 100 ]. The mechanism mediated by Tetra-Py(+)-Me (Fig. 2 ) [ 12 ] was also evaluated. ...
... It was concluded that O (type II mechanism) played the most important role in the aPDT process mediated by the cationic porphyrins Tri-Py(+)-Me, Tetra-Py(+)-Me, and Tri-Spy(+)-Me-PF. Nevertheless, a contribution of the type I pathway cannot be neglected, mainly when Tri-Spy(+)-Me-PF was used; a protection of 18% occurred in the presence of the free radical scavenger -mannitol [ 100 ]. ...
The emergence of microbial resistance to antimicrobials among several common pathogenic microbial strains is an increasing problem worldwide. Thus, it is urgent to develop not only new antimicrobial therapeutics to fight microbial infections, but also new effective, rapid, and inexpensive methods to monitor the efficacy of these new therapeutics. Antimicrobial photodynamic therapy (aPDT) and antimicrobial blue light (aBL) therapy are receiving considerable attention for their antimicrobial potential and represent realistic alternatives to antibiotics. To monitor the photoinactivation process provided by aPDT and aBL, faster and more effective methods are required instead of laborious conventional plating and overnight incubation procedures. Bioluminescent microbial models are very interesting in this context. Light emission from bioluminescent microorganisms is a highly sensitive indication of their metabolic activity and can be used to monitor, in real time, the effects of antimicrobial agents and therapeutics. This chapter reviews the efforts of the scientific community concerning the development of in vitro, ex vivo, and in vivo bioluminescent bacterial models and their potential to evaluate the efficiency of aPDT and aBL in the inactivation of bacteria.
... PDT has adequacy against a broad spectrum of Gram-positive and Gramnegative bacteria and other types of micro-organisms. It has a multi-target mechanism [26][27][28][29][30], autonomously affecting their protection from standard antimicrobial treatment [31][32]. This Conversely, no enzyme can detoxify •OH or 1 O 2 , making them intensely cytotoxic [34]. ...
... Because if the number of viable bacteria in each sample decreases due to these thermal effects, it can affect the enzyme activity in the results [32]. Our results show an increase in the number of bacterial colonies after two minutes of laser irradiation alone ( Figure 2). ...
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
MgCa and MgGd series of alloys are often reported as promising candidates for biomedical applications. In the present study, cytotoxicity and corrosion behavior of Mg1Ca and Mg10Gd alloys in different electrolytes (NaCl, PBS, MEM) have been investigated in order to make a direct comparison and understand the mechanisms behind their performance. Potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) were employed to analyze corrosion processes depending on media composition, whereas X-Ray diffraction (XRD) and scanning electron microscopy (SEM) were used to evaluate crystalline structure, phase composition and surface morphology of the corroded substrates after immersion in the different electrolytes. Moreover, cytotoxicity of the Mg alloys was assessed using the WST-1 reduction and lactate dehydrogenase (LDH) release assays in L929 mouse fibroblasts. The electrochemical results showed that Mg1Ca has a lower degradation rate when compared to Mg10Gd, due to the lower microgalvanic effects and the presence of Ca as an alloying element. Furthermore, the corrosion activity is reduced in MEM, for both alloys, when compared to NaCl and PBS. The cytotoxicity assays revealed that Mg10Gd was cytotoxic in all the conditions tested, while the toxicity of Mg1Ca was low. Overall, these findings show that Mg1Ca alloy presents a higher corrosion resistance and biocompatibility and is a promising material to be used in biomedical implants.
... In efforts to develop alternative approaches to overcome the deficiencies encountered in conventional antimicrobial treatments in several areas, PDT (Table S3) has been tested by several researchers in the control of pathogenic microorganisms (Luksiene et al. 2004;Tavares et al. 2011;Alves et al. 2014Alves et al. , 2015Simões et al. 2016;Lopes et al. 2020). ...
... Antimicrobial PDT (aPDT) consists in the use of highly cytotoxic reactive oxygen species (ROS), resulting from the combination of photosensitizer molecules, light and molecular oxygen (Alves et al. 2014), that interacts with the biological components of the external structures of microorganisms. Due to the way this antimicrobial treatment works, microorganisms cannot develop resistance against aPDT (Tavares et al. 2010(Tavares et al. , 2011Bartolomeu et al. 2016Bartolomeu et al. , 2017Bartolomeu et al. , 2018Coimbra et al. 2016;Jesus et al. 2018). ...
... In efforts to develop alternative approaches to overcome the deficiencies encountered in conventional antimicrobial treatments in several areas, PDT (Table S3) has been tested by several researchers in the control of pathogenic microorganisms (Luksiene et al. 2004;Tavares et al. 2011;Alves et al. 2014Alves et al. , 2015Simões et al. 2016;Lopes et al. 2020). ...
... Antimicrobial PDT (aPDT) consists in the use of highly cytotoxic reactive oxygen species (ROS), resulting from the combination of photosensitizer molecules, light and molecular oxygen (Alves et al. 2014), that interacts with the biological components of the external structures of microorganisms. Due to the way this antimicrobial treatment works, microorganisms cannot develop resistance against aPDT (Tavares et al. 2010(Tavares et al. , 2011Bartolomeu et al. 2016Bartolomeu et al. , 2017Bartolomeu et al. , 2018Coimbra et al. 2016;Jesus et al. 2018). ...
Main conclusion
Phage-based biocontrol strategies can be an effective alternative to control Psa-induced bacterial canker of kiwifruit.
Abstract
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.
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
Zinc(II) phthalocyanines (Pcs) peripherally decorated with 2,4,6-tris(dimethylaminomethyl)phenoxy groups (Pcs 1 and 3) and the corresponding quaternized derivatives (Pcs 2 and 4) were synthesized and their photodynamic inactivation (PDI) efficiency against a recombinant bioluminescent Escherichia coli was examined. The photophysical data revealed that the presence of the ammonium units on the Pc structures promote a red shift of the absorption bands when compared with the corresponding non-quaternized ones. The ammonium-substituted Pcs 2 and 4 showed excellent stability in DMF, moderate photostability and interesting efficiency to generate singlet oxygen (¹O2). The water-soluble photosensitisers 2 and 4 at 5.0 µM exhibited a high PDI efficiency against planktonic bioluminescent E. coli reaching the detection limit of the methodology (a decrease of ~4 log in the bioluminescence signal) after 210 min and 150 min under red light, delivered at a fluence rate of 135 mW.cm–2, respectively. Moreover, and for the first time, it was accessed the combined action of KI with ammonium-substituted Pcs. The addition of potassium iodide improved significantly the efficacy of Pc 2, that could reach the same inactivation rate after a short period of 5 min upon the same irradiation conditions. The use of KI potentiates the PDI efficacy probably due to the generation of additional highly cytotoxic species during the photodynamic process which begins with the reaction of ¹O2 with KI producing peroxyiodide species. The results of this work show that Pcs 2 and 4, with or without KI, can be considered as promising Pc dyes for the PDI of Gram-negative bacteria.
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
Picado, A.; Oliveira, V.; Pereira, H.; Sousa, M.C.; Costa, L.; Almeida, A., and Dias, J.M., 2020. Assessing the potential of Minho and Lima estuaries for aquaculture. In: Malvárez, G. and Navas, F. (eds.), Global Coastal Issues of 2020. Journal of Coastal Research, Special Issue No. 95, pp. 148-152. Coconut Creek (Florida), ISSN 0749-0208.
This work aims the identification of the best potential areas to develop aquaculture activities in Minho and Lima estuaries, especially for the species with the highest commercial value. The Analytic Hierarchy Process (AHP) methodology was applied to the results of an annual simulation with Delft3D model (including the hydrodynamic and water quality modules). After, each variable used in AHP was crossed with the environmental thresholds for development/growth of one bivalve (Portuguese Oyster) and one fish (Gilthead Seabream) species. In this way, an Exploration Index (EI) was defined in order to identify the most suitable regions according to water biophysical properties. This EI was mapped for both estuaries and results show that, in general, the low and middle estuaries present the best conditions for aquaculture development of bivalve and fish. In addition, the upper areas of both estuaries are unsuitable for aquaculture, due to the low salinity of these regions.
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
Porphyrins (Pors) are well-known photoactive molecules with recognized features to be explored as photosensitisers in the photodynamic inactivation (PDI) of microorganisms. The use of appropriate moieties like pyridinium groups is a remarkable strategy to add peripheral and internal positive charges in the Por structure making them more effective PSs against Gram-negative bacteria like Escherichia coli (E. coli). In this context, an easy synthetic access to obtain cationic Zn(II) porphyrins bearing thiopyridinium and inverted methoxypyridinium units (ZnPors 1c and 2c) was developed and their photo-physical and photo-chemical properties were evaluated. The photodynamic effectiveness of these complexes against a E. coli strain was also studied and compared with the efficacy of the corresponding free-bases (Pors 1b and 2b) and of the neutral precursors (Pors 1, 1a, 2, 2a), these last ones in the absence and in the presence of potassium iodide salt (KI). The obtained results demonstrate high PDI efficiency with the cationic free-base 1b and the ZnPor 2c; both derivatives were able to photoinactivate E. coli till the detection limit of the method at a concentration of 1.0 μM after 20 and 15 min of white light irradiation (25 mW cm⁻²), respectively. Interestingly, under the same experimental conditions (1.0 μM and white light), the neutral Pors (1, 1a, 2 and 2a) with addition of KI salt show a fast inactivation rate, reaching the detection limit of the method after 5 min of irradiation.
... Other aspect to be considered when establishing an optimal aPDT protocol is the PS mechanism of action [20,21]. It is important to evaluate the type of ROS produced during aPDT in order to determine the suitable conditions in which microbial photosensitization should operate and to design improved PS molecules. ...
... It is important to evaluate the type of ROS produced during aPDT in order to determine the suitable conditions in which microbial photosensitization should operate and to design improved PS molecules. Although it is well established that the efficiency of aPDT is related to the ability of a PS to generate 1 O 2 (type II mechanism) and/or free radical species (type I mechanism) in the course of the photodynamic process [20,21], the possibility of oxygen-independent photoinactivation leading to the killing of pathogenic microorganisms, which may be termed the "Type III photochemical pathway", has also been suggested [34]. The proposed mechanism involves photoinduced electron transfer that produces reactive inorganic radicals, which might be useful to treat anaerobic infections or infections in hypoxic tissues [34]. ...
The growing emergence of microbial resistance to conventional antimicrobials, due their dissemination in the environment, and excessive or inadequate prescriptions, associated with the globalization of pathogenic microorganisms’ transmission, make the discovery of new effective therapies to combat infection of extreme urgency [...]
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
In this study, brewers' spent grain (BSG) arabinoxylans-based nanocomposite films were prepared by solvent casting of arabinoxylans (AX) suspensions containing different amounts of nanofibrillated cellulose (NFC, 5, 10, 25, 50 and 75% mass fraction). The obtained nanocomposite films were homogeneous and presented thermal stability up to 230 °C and good mechanical properties (Young's modulus up to 7.5 GPa). Additionally, the films with 50% NFC were loaded with ferulic acid or feruloylated arabinoxylo-oligosaccharides enriched fraction from BSG (75 mg per g of film). This combination enhanced the UV–Vis barrier properties and imparted additional functionalities to the films, namely (i) antioxidant activity up to 90% (DPPH scavenging activity), (ii) antibacterial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, and (iii) antifungal activity towards the polymorphic fungus Candida albicans. Therefore, these fully biobased nanocomposite films show potential for application as active food packaging systems.
... Therefore, efforts should be made towards the development of more efficient, non-toxic, and noninvasive antimicrobial methods to apply to the hosts. Importantly, these new methods should not induce the development of antimicrobial resistance [15][16][17]. Toward this end, antimicrobial photodynamic therapy (aPDT) has been considered as a promising non-antibiotic approach to inactivate foodborne bacteria [18][19][20][21][22][23]. ...
... These ROS can react with biological molecules (e.g., proteins, lipids, and nucleic acids) causing microbial death [16,24,25]. This technique presents several advantages when compared with the use of traditional antimicrobials, showing to be efficient independently of the antimicrobial resistance profile and to prevent further development of resistance even after several cycles of treatment [15][16][17]26]. This approach has been efficient to inactivate several microorganisms, such as gram-negative and gram-positive bacteria [18,19,21], fungi [15,[27][28][29], and viruses [15,30], and to degrade the matrix of microbial biofilms and kill the resident bacteria [16,31,32]. ...
Antimicrobial photodynamic therapy (aPDT) has been shown as a promising technique to inactivate foodborne bacteria, without inducing the development of bacterial resistance. Knowing that addition of inorganic salts, such as potassium iodide (KI), can modulate the photodynamic action of the photosensitizer (PS), we report in this study the antimicrobial effect of eosin (EOS) and rose bengal (RB) combined with KI against Salmonella enterica serovar Typhimurium and Staphylococcus aureus. Additionally, the possible development of bacterial resistance after this combined aPDT protocol was evaluated. The combination of EOS or RB, at all tested concentrations, with KI at 100 mM, was able to efficiently inactivate S. Typhimurium and S. aureus. This combined approach allows a reduction in the PS concentration up to 1000 times, even against one of the most common foodborne pathogenics, S. Typhimurium, a gram-negative bacterium which is not so prone to inactivation with xanthene dyes when used alone. The photoinactivation of S. Typhimurium and S. aureus by both xanthenes with KI did not induce the development of resistance. The low price of the xanthene dyes, the non-toxic nature of KI, and the possibility of reducing the PS concentration show that this technology has potential to be easily transposed to the food industry.
... nucleic acids, proteins and lipids) causing microbial death. [3][4][5][6] aPDT presents several advantages when compared with the use of traditional antimicrobials. This approach proves to be efficient and independent of the antimicrobial resistance profile, 5 and is able to prevent further development of resistance even after several cycles of treatment. ...
... Before each assay, one isolated colony was transferred to 10 mL of Luria Bertani medium (Liofilchem, Italy) previously supplemented with Amp and Cm and was grown overnight at 37°C under stirring (120 rpm). An aliquot was transferred into 10 mL Tryptic Soy Broth (TSB, Merck) under the same growth conditions till the stationary growth phase was achieved [≈10 6 relative light unit (RLU)]. An optical density of 1.6 ± 0.1 at 600 nm (OD600) corresponded to ≈10 6 colony forming units (CFU) per mL. ...
New porphyrin/4-oxoquinoline conjugates were synthesized from the Heck coupling reaction of a β-brominated porphyrin with 1-allyl-4-oxoquinoline derivatives, followed by demetallation and deprotection affording the promissing photossensitizers 9a-e. The singlet oxygen studies have demonstrated that all the porphyrin/4-oxoquinoline conjugates 9a-e were capable to produce that cytotoxic species and revealed to be excellent photossensitizer agents in the inactivation of S. aureus after the antimicrobial photodynamic therapy (aPDT) protocol.
... Tavares et al. [99] examined the mechanisms of photodynamic inactivation (PDI) of bacteria using cationic porphyrins. They proposed that singlet oxygen is more responsible for the PDI process of the bioluminescent E. coli than free radicals generated by the cationic porphyrins. ...
... aPDT is independent towards the resistance pattern of bacteria to antibiotics; it produces extensive bacteria reduction with restricted damage to healthy host tissue, and specifically delivers PS to the infected area [81,83]. It demonstrates fast inactivation than usual antimicrobials [11,99,109,110]. It shows absence of photo-resistant strains and viability recovery after multiple treatments [80,[111][112][113]. ...
Antibiotics are commonly used to control, treat, or prevent bacterial infections, however bacterial resistance to all known classes of traditional antibiotics has greatly increased in the past years especially in hospitals rendering certain therapies ineffective. To limit this emerging public health problem, there is a need to develop non-incursive, non-toxic, and new antimicrobial techniques that act more effectively and quicker than the current antibiotics. One of these effective techniques is antibacterial photodynamic therapy (aPDT). This review focuses on the application of porphyrins in the photo-inactivation of bacteria. Mechanisms of bacterial resistance and some of the current ‘greener’ methods of synthesis of meso-phenyl porphyrins are discussed. In addition, significance and limitations of aPDT are also discussed. Furthermore, we also elaborate on the current clinical applications and the future perspectives and directions of this non-antibiotic therapeutic strategy in combating infectious diseases.
... The efficacy of the photodynamic action of P2, P3 and P4 and of the new materials was assessed in the presence of recombinant bioluminescent E. coli. This Gram-negative bacterium is an excellent model to monitor the effectiveness of a photoinactivation process, since its light output is a sensitive sign of its metabolic activity [16,19,20,[52][53][54][55]. Additionally, E. coli is one of the most common Gram-negative pathogens in humans, causing several serious illnesses, and it is known to develop multidrug resistance [38,56]. ...
... It is commonly accepted that the success of photoinactivation closely depends on the structure of the photosensitizing agent [14]. As previously stated, the charge of the PS is extremely important for the inactivation of the microbial agents and, generally, positively charged PS are more efficient for Gram-negative strains, even if they are at low concentrations when compared to neutral or negatively charged PS [52]. Studies with cationic PS demonstrate that these derivatives are more likely to inactivate both Gram-positive and Gram-negative bacteria without the help of membrane disrupting agents [47,76]. ...
The post-functionalization of 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide, known as a highly efficient photosensitizer (PS) for antimicrobial photodynamic therapy (aPDT), in the presence of 3- or 4-mercaptobenzoic acid, afforded two new tricationic porphyrins with adequate carboxylic pending groups to be immobilized on chitosan or titanium oxide. The structural characterization of the newly obtained materials confirmed the success of the porphyrin immobilization on the solid supports. The photophysical properties and the antimicrobial photodynamic efficacy of the non-immobilized porphyrins and of the new conjugates were evaluated. The results showed that the position of the carboxyl group in the mercapto units or the absence of these substituents in the porphyrin core could modulate the action of the photosensitizer towards the bioluminescent Gram-negative Escherichia coli bacterium. The antimicrobial activity was also influenced by the interaction between the photosensitizer and the type of support (chitosan or titanium dioxide). The new cationic porphyrins and some of the materials were shown to be very stable in PBS and effective in the photoinactivation of E. coli bacterium. The physicochemical properties of TiO2 allowed the interaction of the PS with its surface, increasing the absorption profile of TiO2, which enables the use of visible light, inactivating the bacteria more efficiently than the corresponding PS immobilized on chitosan.
... The ratio of both processes is described by the singlet oxygen quantum yield Φ Δ which is unique for each PS and depends on the respective chemical structure. Particularly, PS that mainly acts according to type II mechanism such as the fourfold positively charged porphyrin derivative TMPyP [5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)-21,23Hporphine tetratosylate)] has shown promising antibacterial efficacy in vitro [11][12][13][14][15]. ...
The widespread occurrence of multi-resistant bacteria is a health problem of global dimension. Infections caused by multi-resistant pathogens are difficult to treat and often associated with high mortality. Therefore, new treatment strategies are of interest, such as the use of differently acting antibacterial concepts. One of these new concepts is the use of antiseptics in combination with the antibacterial photodynamic therapy (aPDT). Currently, no method has yet been established as a standard procedure for investigating combined effects and evaluating them in a generally valid and unambiguous manner. The focus of this study was on how cationic antiseptics benzalkonium chloride (BAC) and chlorhexidine digluconate (CHX) behave in a combined application with aPDT using the photosensitizer TMPyP. For this purpose, BAC and CHX were applied in combination with the aPDT using TMPyP in non-lethal concentrations to the three bacteria Escherichia coli , Staphylococcus aureus , and Enterococcus faecalis . The results of the combination experiments with sublethal concentrations of BAC or CHX with the aPDT showed that the binary application had a lethal effect. Irrespective of the bacteria, the reduction in concentrations in OPECC, compared to individual concentrations, was more than 50% for TMPyP, 23–40% for BAC, and 18–43% for CHX. Furthermore, the optimal effective concentration combinations (OPECCs) could be determined. The latter showed that the combined application allowed the reduction of both concentrations compared to the single application.
... At the same time, cationic PSs can also target the suborganelles of cancer cells, which can promote a series of inammatory and immune responses by the ROS produced during PDT, destroy the tumor microvasculature, and ultimately induce tumor cell death. [26][27][28] However, cationic PSs can still be induced to aggregate by the physiological environment, biological polyanions (e.g., adenosine triphosphates (ATP) and pyrophosphate (PPi)), and high ionic strength, resulting in serious uorescence quenching which greatly limits the potential in vivo applications. 7,29,30 Therefore, it is very promising to develop cationic molecular PSs with excellent anti-ACQ properties in a physiological environment. ...
Non-AIE-type molecular photosensitizers (PSs) suffer from the aggregation-caused-quenching (ACQ) effect in an aqueous medium due to the strong hydrophobic and π–π interactions of their conjugated planes, which significantly hinders the enhancement of tumor photodynamic therapy (PDT). So far, some ionic PSs have been reported with good water-solubility, though the ACQ effect can still be induced in a biological environment rich in ions, leading to unsatisfactory in vivo delivery and fluorescence imaging performance. Hence, designing molecular PSs with outstanding anti-ACQ properties in water is highly desirable, but it remains a tough challenge for non-AIE-type fluorophores. Herein, we demonstrated a strategy for the design of porphyrin-type molecular PSs with remarkable solubility and anti-ACQ properties in an aqueous medium, which was assisted by quantum chemical simulations. It was found that cationic branched side chains can induce serious plane distortion in diphenyl porphyrin (DPP), which was not observed for tetraphenyl porphyrin (TPP) with the same side chains. Moreover, the hydrophilicity of the chain spacer is also crucial to the plane distortion for attaining the desired anti-ACQ properties. Compared to ACQ porphyrin, anti-ACQ porphyrin displayed type-I ROS generation in hypoxia and much higher tumor accumulation efficacy by blood circulation, leading to highly efficient in vivo PDT for hypoxic tumors. This study demonstrates the power of sidechain chemistry in tuning the configuration and aggregation behaviors of porphyrins in water, offering a new path to boost the performance of PSs to fulfill the increasing clinical demands on cancer theranostics.
... In the search for alternative strategies to treat multidrug-resistant skin infections, antimicrobial photodynamic therapy (aPDT) has proven to be a promising option [4]. This approach uses light with appropriate wavelengths, along with a photosensitizing agent, which in the presence of molecular oxygen generates oxidant species [reactive oxygen species (ROS) like superoxide and singlet oxygen, ( 1 O 2 )], that kill pathogens, including multidrug resistant bacteria [5]. ...
Antimicrobial photodynamic therapy (aPDT) is a potent tool to surpass the global rise of antimicrobial resistance; still, the effective topical administration of photosensitizers remains a challenge. Biopolymer-based adhesive films can safely extend the residence time of photosensitizers. However, their wide application is narrowed by their limited water absorption capacity and gel strength. In this study, pullulan-based films with a switchable character (from a solid film to an adhesive hydrogel) were developed. This was accomplished by the incorporation of a betaine-based deep eutectic solvent (DES) containing curcumin (4.4 μg.cm-2) into the pullulan films, which tuned the films' skin moisture absorption ability, and therefore they switch into an adhesive hydrogel capable of delivering the photosensitizer. The obtained transparent films presented higher extensibility (elongation at break up to 338.2%) than the pullulan counterparts (6.08%), when stored at 54% of relative humidity, and the corresponding hydrogels a 4-fold higher adhesiveness than commercial hydrogels. These non-cytotoxic adhesives allowed the inactivation (∼5 log reduction), down to the detection limit of the method, of multiresistant strains of Staphylococcus aureus in ex vivo skin samples. Overall, these materials are promising for aPDT in the treatment of resistant skin infections, while being easily removed from the skin.
... The use of scavengers helps in identifying the relative importance of the two pathways. The study concluded that photoinactivation of the bioluminescent E. coli by Tri-Py (+)-Me-PF, Tetra-Py(+)-Me and Tri-SPy(+)-Me-PF occurs mainly through singlet oxygen (type II) than free radicals (type I) mechanism (Tavares et al., 2011). These reactions result in functional damage, morphological changes and cell membrane damage in bacteria. ...
Resistance to antimicrobial drugs is an impending healthcare problem of growing significance. In the post-antibiotic era, there is a huge push to develop new tools for effectively treating bacterial infections. Photodynamic therapy involves the use of a photosensitizer that is activated by the use of light of an appropriate wavelength in the presence of oxygen. This results in the generation of singlet oxygen molecules that can kill the target cells, including cancerous cells and microbial cells. Photodynamic therapy is shown to be effective against parasites, viruses, algae, and bacteria. To achieve high antimicrobial activity, a sufficient concentration of photosensitizer should enter the microbial cells. Generally, photosensitizers tend to aggregate in aqueous environments resulting in the weakening of photochemical activity and lowering their uptake into cells. Nanocarrier systems are shown to be efficient in targeting photosensitizers into microbial cells and improve their therapeutic efficiency by enhancing the internalization of photosensitizers into microbial cells. This review aims to highlight the basic principles of photodynamic therapy with a special emphasis on the use of nanosystems in delivering photosensitizers for improving antimicrobial photodynamic therapy.
... These cytotoxic species can then damage cellular components such as proteins, membrane fatty acids, amino acid residues and nucleotide bases, leading to loss of organic and metabolic function of the cell (Wainwright et al., 2017). Studies have already demonstrated the effectiveness of this method against bacteria, including some multidrug-resistant strains, fungi, spores, yeasts, protozoa, viruses and biofilms, without detection, so far, of resistance or mutagenicity after treatment (Alves et al., 2013;Beirão et al., 2014;de Menezes et al., 2014;Tavares et al., 2011). Despite PDI being widely studied in human medicine in recent years, very little of this methodology has been explored for the control of phytopathogens. ...
Grain production is of great importance in the world economy, with soybeans being the main agricultural product. Among the main causes of crop losses are diseases caused by pathogens that infect seeds, such as Colletotrichum truncatum, Corynespora cassiicola, Sclerotinia sclerotiorum and Rhizoctonia solani. The fungicides used are not fully effective in controlling these diseases, and new control options are necessary. Photodynamic Inactivation (PDI) may be an alternative. PDI consists of the association of photosensitizer (PS), light and molecular oxygen aiming at the destruction of microbial cells. Porphyrins and their derivatives are aromatic tetrapyrrolic macrocyclic compounds with photosensitizing capacity. The aim of the present study was to evaluate the efficiency of PDI in the photoinactivation of C. truncatum, C. cassiicola, S. sclerotiorum and R. solani, using a mixture of cationic meso‐tetra‐aryl porphyrins as PS, being the first study to consider the formulation tested as PS in the photoinactivation of these pathogens. Assays were carried out with soybean seeds artificially inoculated with the different fungi, treated with the PS at 10, 50, 100 and 200 μg.mL‐1 and submitted to light under an irradiance of 50 mW.cm‐2. The assays were set up following the “Blotter test” methodology. The evaluations showed significant reductions in the infection rates of the seeds in relation to the controls. The greatest reductions were 46% for C. truncatum, 95.5% for S. sclerotiorum, 87% for C. cassiicola and 92% for R. solani. The results suggest that PDI can be an important alternative to current control methods against these pathogens.
... Cationic porphyrins show their antimicrobial activity through the type II reaction mechanism. Single oxygen (¹O₂) formed by the transfer of the energies of light-induced cationic porphyrins to molecular oxygen reacts with structures such as phospholipids, peptides, and sterols in the cell wall and cell membrane and causes cell death [11][12][13][14]. In our previous study, a strong aPDI was obtained on methicillin-resistant Staphylococcus aureus (MRSA) [15]. ...
Antimicrobial photodynamic inactivation (aPDI) of multidrug-resistant (MDR) wound pathogens was evaluated with cationic porphyrin derivatives (CPDs). MDR bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae were used. The CPDs named PM, PE, PN, and PL were synthesized as a photosensitizer (PS). A diode laser with a wavelength of 655 nm was used as a light source. aPDI of the combinations formed with different energy densities (50, 100, and 150 J/cm²) and PS concentrations (ranging from 3.125 to 600 µM) were evaluated on each bacterial strain. Dark toxicity, cytotoxicity, and phototoxicity were determined on fibroblast cells. In the aPDI groups, survival reductions of up to 5.80 log₁₀ for E. coli, 5.90 log₁₀ for P. aeruginosa, 6.11 log₁₀ for K. pneumoniae, and 6.78 log₁₀ for A. baumannii were obtained. The cytotoxic effect of PL and PM on fibroblast cells was very limited. PN was the type of CPD with the highest dark toxicity on fibroblast cells. In terms of providing broad-spectrum aPDI without or with very limited cytotoxic effect, the best result was observed in aPDI application with PL. The other CPDs need some modifications to show bacterial selectivity for use at 50 µM and above.
... The type of ROS produced vary in proportion between oxygen radicals, such as superoxide (O 2 x ‾) or hydroxyl (OH x ), and singlet oxygen ( 1 O 2 ) depending on the photosensitiser's characteristics [2]. Because 1 O 2 appears to account for most of the antimicrobial effect exerted by aPDT, tetrapyrrolic porphyrinic photosensitisers that display high quantum yields of 1 O 2 , such as TMPyP, are commonly employed [3,4]. The stress imposed by 1 O 2 is termed photo-oxidative stress, as this ROS is almost exclusively generated by light-dependent processes [5,6]. ...
Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes.
Six Enterococcus faecalis strains were investigated; ATCC-29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR.
TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 μM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma-factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma-factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015).
These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.
... In this study, however, no damage was observed on leaves treated with photoactivated RF. It is well documented that when PS is applied at low concentrations and for short periods, aPDT can be specific to microorganisms (Tavares et al. 2011;Beirão et al. 2014). Leaf microtextured may also serve as a barrier, stopping PS from crossing the leaf cuticle. ...
Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv oryzae (Xoo), is one of the most damaging rice diseases, causing severe production losses depending on the rice variety. The purpose of this study was to develop an antibacterial photodynamic treatment (aPDT) using riboflavin for the treatment of BLB disease. Combining light and riboflavin (RF) therapy significantly reduced bacterial planktonic cells compared to RF alone. Photoactivated riboflavin also decreased biofilm biomass by reducing the number of viable sessile cells and the production of extracellular polymeric substances (EPS). Reactive oxygen species (ROS) levels in Xoo cells treated with photoactivated riboflavin were found to be significantly higher than in cells treated with riboflavin and light individually. Malondialdehyde (MDA) increased greatly in photoactivated riboflavin treated cells, indicating that severe oxidative damage was induced. Subsequently, a reduction in lactate dehydrogenase (LDH) activity in photoactivated riboflavin treated Xoo cells indicates that oxidative stress has disrupted the respiratory system, leading to bacterial cell death. In an ex vivo aPDT assay, photoactivated riboflavin successfully eradicated Xoo on the surface of rice leaves. Photoactivated riboflavin had no side effects on rice seed germination in subsequent trials, indicating that it is safe for agricultural applications. Therefore, all these findings suggest that aPDT is a potential alternative management strategy for BLB disease.
Graphical abstract
... Ongoing research focuses on further optimization of photosensitizers for specific applications [12,13], as well as on the in-detail characterization and comparison of different photosensitizers in vitro [14][15][16]. Additionally, photosensitizers were tested in in vivo models for critical infections [17][18][19]. ...
Antimicrobial resistance belongs to the most demanding medical challenges, and antimicrobial photodynamic inactivation (aPDI) is considered a promising alternative to classical antibiotics. However, the pharmacologic characterization of novel compounds suitable for aPDI is a tedious and time-consuming task that usually requires preparation of bacterial cultures and counting of bacterial colonies. In this study, we established and utilized a luminescence-based microbial cell viability assay to analyze the aPDI effects of two porphyrin-based photosensitizers (TMPyP and THPTS) on several bacterial strains with antimicrobial resistance. We demonstrate that after adaptation of the protocol and initial calibration to every specific bacterial strain and photosensitizer, the luminometric method can be used to reliably quantify aPDI effects in most of the analyzed bacterial strains. The interference of photosensitizers with the luminometric readout and the bioluminescence of some bacterial strains were identified as possible confounders. Using this method, we could confirm the susceptibility of several bacterial strains to photodynamic treatment, including extensively drug-resistant pathogens (XDR). In contrast to the conventional culture-based determination of bacterial density, the luminometric assay allowed for a much more time-effective analysis of various treatment conditions. We recommend this luminometric method for high-throughput tasks requiring measurements of bacterial viability in the context of photodynamic treatment approaches.
... These findings match the results of Jadah et al. 30,31 APDT has adequacy against a wide spectrum of gram- It has a great advantage of the multitarget mechanism, 32-39 autonomously their protection from customary antimicrobial treatment. [40][41][42] This method requires a close photosensitizer (PS), light, and oxygen. The PS when energized by laser light within the sight of O 2 produces ROS, which are superoxide (O 2 ...
Introduction: Staphylococcus aureus is one of the critical pathological bacteria. This bacterium had developed a variety of genetic mutations that made it resistant to drugs and more harmful to humans. In addition, all attempts to design a specific vaccine against S. aureus have failed. Therefore, this experiment was designed as a trial for vaccine production, by using a photodynamic treatment (PDT) through partial biological inhibition. The PDT of bacteria mainly focused on reducing the activity of staphylocoagulase (SC), which has a protective feature for bacteria. This study aimed to examine the photodynamic effect of combining a specific wavelength of a laser and a certain dilution photosensitizer, methylene blue (MB) dye. The possible PDT effect on the inhibition of pathogenic enzymatic activity was predicted. This study also aimed to evaluate the inhibitory effect of PDT on the total bacterial account (viability) simultaneously with SC assay. Methods: A 650nm wavelength diode laser was used with 100 mW output power and 2 minutes of exposure time. Dye dilutions were 50, 100, 150 and 200 μg/mL. The viability of bacteria after and before laser treatment was calculated using single plate-serial dilution spotting methods. The activity of SC was detected by using human plasma for 4 hours incubation of crude-substrate interaction. Results: The results revealed a significant decrease in enzyme activity and colony-forming units (CFU) after irradiating bacterial suspension with 150 g/mL MB, as well as a decline in CFU. However, irradiation with a laser alone showed a significant increase in SC activity and CFU for the same exposure time. Conclusion: Besides reducing the production of SC activity, PDT significantly inhibited the viability of S. aureus. The application of MB photosensitizer at a concentration of 150 g/mL in combination with a laser wavelength of 650 nm resulted in a complete decrease in the SC activity value as well as the viability of bacteria.
... A designed PS containing iodine atoms [2,6-diiodo-1,3,5,7-tetramethyl-8-(P-benzoic acid)-4,40-difluoroboradiazaindacene, DIBDP], with phthalocyanine derivatives as coadjutants for improved photostability, was more effective in the control of CCK symptoms in mandarin leaves than copper biocides, with lower concentrations and negligible damage to the leaves (Jiang et al. 2020). Iodine, either added as an adjutant or incorporated in the PS molecule, seems to be important for the photosensitization of Xcc, indicating that, like other Gram-negative bacteria, photosensitization occurs mainly by a Type II mechanism (Tavares et al. 2011). ...
Citrus are economically important fruit crops to which infectious diseases like citrus canker caused by Xanthomonas citri subs. citri, citrus variegated chlorosis caused by Xylella fastidiosa, “huanglongbing” associated with the presence of Candidatus liberibacter species, anthracnose caused by Colletotrichum gloeosporioides and citrus black spot caused by Phyllosticta citricarpa, impose significant losses. Control measures involve chemical treatment of orchards but often, eradication of infected plants is unavoidable. To circumvent the environmental impacts of pesticides and the socio-economic impacts of eradication, innovative antimicrobial approaches like photodynamic inactivation are being tested. There is evidence of the susceptibility of Xanthomonas citri subs. citri and C. gloeosporioides to photodynamic damage. However, the realistic assessment of perspectives for widespread application of photodynamic inactivation in the control of citrus diseases, necessarily implies that other microorganisms are also considered. This review intends to provide a critical summary of the current state of research on photodynamic inactivation of citrus pathogens and to identify some of the current limitations to the widespread use of photodynamic treatments in citrus crops.
... The production of singlet oxygen has been determined using anthracene-9,10-dipropionic acid (ADPA) as selective probe. A 10 µM solution of ADPA was prepared in 0.01 PBS (total volume 3 mL) and irradiated with blue LED, with and without active PVC films (1 cm 2 ), for 90 min, and the decay of the ADPA absorbance at 377 nm was recorded at regular intervals [23,53]. ...
Synthesis, chemical, and biological characterization of composite polyvinyl chloride (PVC)-based materials to be applied in the photodynamic inactivation (PDI) of pathogen microorganisms are reported. Different blends of PVC, plasticizers, and photosensitizers have been tested in their photokilling ability vs. Staphylococcus aureus, after irradiation using a multi-LED blue lamp at a fluence rate of 50 W/m². Four common adipates, namely: dibutyl hexanedioate [di(n-butyl) adipate, BA, 1], bis(2-ethylhexyl) hexanedioate [bis(2-ethylhexyl) adipate, EA, 2], dioctyl hexanedioate (dioctyl adipate, OA, 3), and didecyl hexanedioate (dicapryl adipate, DA, 4), have been employed in this investigation, in combination with two photosensitizers: 5-(4-carboxy-phenyl)-10,15,20-triphenyl-21H,23H-porphyrin (TPP, 5), and the expanded porphyrin 20-(4-carboxyphenyl)-2,13-dimethyl-3,12-diethyl-[21]pentaphyrin (PCox, 6). The experimental evidence has established the essential role of the addition of a plasticizer to the polymer/photosensitizer mixture in transforming PVC into a photoactive material, being bactericidal properties dependent upon the type and amount of additive. Long-chain linear adipates were found more efficient in imparting the desired bactericidal activity to the final material, reaching in one case the complete abatement of the initial bacterial solution (10⁸ CFU/ml) in 60 min. The obtained polymeric films proved to be stable over time and under oxidation conditions; in addition, no release of toxic components was observed over the experiments, definitely demonstrating that the bactericidal action was effectively due to the ROS generated by photosensitizers immobilized into the material. Moreover, SEM and FT-IR analyses proved that no photodegradation of the film took place during the irradiation experiment.
... Therefore, it is very difficult to be able to make a direct extrapolation of the photodynamic properties obtained in solution to the processes that take place in the microbial cells. The mechanisms may depend mainly on the nature of the PS, the polarity of the microenvironment where it is located into the cells, and the substrates that are in the vicinity of the PS [43][44][45]. Among the amino acid residues, Trp is one of the compounds most susceptible to oxidation in proteins [46]. ...
A novel tricationic Zn(II)phthalocyanine derivative, (NCH3)3ZnPc3+, was synthesized by ring expansion reaction of boron(III) [2,9(10),16(17)-trinitrosubphthalocyaninato]chloride. First, the reaction of this subphthalocyanine with 2,3-naphthalenedicarbonitrile and Zn(CH3COO)2 catalyzed by 8-diazabicyclo[5.4.0]undec-7-ene was used to obtain the A3B-type nitrophthalocyanine. After reduction of nitro groups with Na2S and exhaustive methylation of amino groups, (NCH3)3ZnPc3+ was formed in good yields. In addition, the tetracationic analog (NCH3)4ZnPc4+ was synthesized to compare their properties. The absorption and fluorescence spectra showed the Q-bands and the red emission, respectively, which are characteristic of the Zn(II)phthalocyanine derivatives in N,N-dimethylformamide. Furthermore, photodynamic activity sensitized by these compounds was studied in the presence of different molecular probes to sense the formation of reactive oxygen species. (NCH3)3ZnPc3+ efficiently produced singlet molecular oxygen and also it sensitized the formation of superoxide anion radical in the presence of NADH, while the photodynamic activity of (NCH3)4ZnPc4+ was very poor, possibly due to the partial formation of aggregates. Furthermore, the decomposition of L-tryptophan induced by (NCH3)3ZnPc3+ was mainly mediated by a type II mechanism. Antimicrobial photodynamic inactivation sensitized by these phthalocyanines was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans, as representative microbial cells. In cell suspensions, (NCH3)3ZnPc3+ was rapidly bound to microbial cells, showing bioimages with red fluorescence emission. After 5 min of irradiation with visible light, (NCH3)3ZnPc3+ was able to completely eliminate S. aureus, E. coli and C. albicans, using 1.0, 2.5 and 5.0 μM phthalocyanine, respectively. In contrast, a low photoinactivation activity was found with (NCH3)4ZnPc4+ as a photosensitizer. Therefore, the amphiphilic tricationic phthalocyanine (NCH3)3ZnPc3+ is a promising photosensitizing structure for application as a broad-spectrum antimicrobial phototherapeutic agent.
... As a consequence, in such a heterogeneous system, more ROS photoproduction can occur, resulting in a higher inactivation rate. As for these PSs, the most predominant pathway in their photodynamic action is via energy transfer [57,92,97] and taking into account the experimental conditions in which the assays were performed (light irradiance, time exposure of the samples to irradiation, incubation temperature), a close interaction between 3 PS * and organic matter, resulting in electron transfer, must not have caused a significant depletion of PS via chemical reaction and (photo)degradation of PS [27,98]. In this study, when the content of particulate organic matter in the aqueous matrix was reduced, the aPDT efficiency had a slight increase. ...
Pathogenic viruses are frequently present in marine and estuarine waters, due to poor wastewater (WW) treatments, which consequently affect water quality and human health. Chlorination, one of the most common methods used to ensure microbiological safety in tertiarily treated effluents, may lead to the formation of toxic chemical disinfection by-products on reaction with organic matter present in the effluents. Antimicrobial photodynamic therapy (aPDT) can be a promising disinfecting approach for the inactivation of pathogens, without the formation of known toxic by-products. Additionally, some studies have reported the potentiator effect on aPDT of some compounds, such as potassium iodide (KI) and hydrogen peroxide (H2O2). In the present study, the aPDT efficiency of a PS formulation constituted of five cationic porphyrins (Form) in the inactivation of E. coli T4-like bacteriophage, a model of mammalian viruses, in different aqueous matrices with different organic matter content, was evaluated. Photoinactivation studies were performed at different concentrations of Form and in the presence of the adjuvants KI and H2O2. The results showed that the efficiency of bacteriophage photoinactivation is correlated with the Form concentration, the amount of the organic matter in WW, and the adjuvant type. Form can be an effective alternative to controlling viruses in WW, particularly if combined with H2O2, allowing to significantly reduce PS concentration and treatment time. When combined with KI, the Form is less effective in inactivating T4-like bacteriophage in WW.
... Sample and LC were irradiated under white light at an irradiance of 50 mW cm −2 for 90 min; the DC was kept in the dark for the same period of aPDT treatment. Aliquots of the sample, LC, and DC were collected at time 0 (after the pre-incubation period and immediately before irradiation) and after 5,15,30,45,60, and 90 min of irradiation, serially diluted in sterile PBS and 3 droplets of 5.0 µL of each dilution were plated in LA. The plates were then incubated at 25 • C for 48 h and the number of CFU was counted on the most appropriate dilution on the agar plates. ...
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.
... Antimicrobial photodynamic therapy (aPDT) seems to be a very promising approach for localized infections in response to the ever-growing problem of antibiotic resistance, including the ones caused by S. aureus [19][20][21][22][23][24]. aPDT has already demonstrated its effectiveness against a wide range of microorganisms like Gram-positive and Gram-negative bacteria, viruses, fungi and parasites [5,21,[25][26][27][28][29][30][31][32]. This therapeutic approach requires the presence of a photosensitizer (PS), light and oxygen; the PS when excited by light produces reactive oxygen species (ROS) that are highly cytotoxic. ...
Staphylococcus aureus is responsible for skin and soft tissue infections. Having in mind the increased antibiotic resistance, in this study the efficacy of antimicrobial photodynamic therapy (aPDT) with a porphyrinic formulation (FORM) as photosensitizer (PS) to photoinactivate methicillin-resistant Staphylococcus aureus (MRSA) on skin was evaluated. Potassium iodide (KI) and iodopovidone (PVP-I) were also tested in combination with FORM as potentiator agents of FORM efficacy. The aPDT protocol was first developed in Phosphate Buffered Saline (PBS, in vitro). Porcine skin was artificially contaminated with MRSA (ex vivo) and treated with FORM, FORM + KI or FORM + PVP-I under white light. The in vitro results showed that FORM was effective to inactivate MRSA. A substantial reduction in the irradiation time, when compared to FORM alone, was observed for FORM + KI and FORM + PVP-I combinations. On skin, reductions in MRSA survival of 3.1 Log10 colony forming units (CFU) mL⁻¹ were observed with FORM at 50 μM. Although the combined action of FORM + KI and FORM + PVP-I potentiated the aPDT efficacy in vitro, this was not observed ex vivo. Overall, the results showed that aPDT using FORM, even without coadjutants, is a promising approach for MRSA inactivation on skin.
... Photodynamic effect is the generation of singlet-oxygen and other reactive oxygen species (ROS) by interaction of a photosensitizer, light of appropriate wavelength and molecular oxygen (Costa et al., 2013;Komagoe, Kato, Inoue, & Katsu, 2011;Lukšiene, 2005;Tavares et al., 2011). Photodynamic sanitation of foods is a process in which the generated singlet-oxygen and other ROS inactivate pathogens and reduces microbial load of the food to a level considered safe by the public health standards Lukšiene, 2005;Luksiene & Brovko, 2013). ...
Photodynamic sanitation of fresh produce could help reduce spoilage and disease transmissions where conventional methods of sanitation are not available, and sunlight is available for free. In this study, we evaluated the photostability and photodynamic antibacterial activity of the dye extracts of calyces of Hibiscus sabdariffa. The dye extracts were very photostable in water but bleached in acetate‐HCl buffer (pH 4.6), phosphate buffer saline (pH 7.2), and tris base‐HCl buffer (pH 8.6). The photostability correlated with the photodynamic antibacterial activity of the dye extracts. Both the methanol and water dye extracts at the concentration of 0.0625 mg/ml caused complete inactivation of Bacillus subtilis (reductions of 8.5 log CFU/ml) within 2 min either with the visible light exposure at 10 mW/cm² or in the dark without the light exposure. Reductions of 4.8 log CFU/ml and 2.2 log CFU/ml of Escherichia coli were observed when 1 mg/ml of methanol and water dye extracts were used, respectively, in water with the light exposure at 10 mW/cm² for 20 min. Discussions are included about the ease of the dye extractions of the calyces of H. sabdariffa even in water without the need of energy for heating and the suitability of the dye extracts for the fresh produce sanitation. Dye extract of calyces of H. sabdariffa has photodynamic and nonphotodynamic antibacterial activity which could be exploited for the development of a low‐tech sunlight‐driven fresh produce sanitation system that is cheap, sustainable, and environmentally friendly.
... [18] Thus, to investigate the underlying mechanism of the bacterial inactivation and find out whether aPDT efficacy is affectedb yt he nature and persistence of the ROS two different quenchers, sodium azide and mannitol, were used in irradiation experiments. [19] Althoughn either of these two scavengers could provide complete protection from inactivation, both sodium azide and mannitol hada ne ffect on the viability of microorganisms. Comparison of the photodynamic effect of Pd-NDP for S. aureus 3150/12 and B. subtilis DB104 in the presence of sodium azide showedt hat the mode of action of Pd-NDP is most likely different for these two strains. ...
Free base, zinc and palladium π‐extended porphyrins containing fused naphthalenediamide units were employed as photosensitizers in antimicrobial photodynamic therapy (aPDT). Their efficacy, assessed by photophysical and in vitro photobiological studies on Gram‐positive bacteria, was found to depend on metal coordination, showing a dramatic enhancement of photosensitizing activity for the palladium complex.
... Six hours of irradiation using 20 μM of TMPyP generated enough ROS (mainly 1 O 2 ) to result in 100% lethality of the model bacterium V. campbellii ISO7 in mixed microalgabacteria incubation experiments. In addition, the DNA of the model bacterium was not detected using molecular techniques; therefore, as DNA is not the primary target of 1 O 2 (Bonnett and Berenbaum 2007;Maisch 2007;Tavares et al. 2011;Dosselli et al. 2012;Sperandio et al. 2013;Almeida et al. 2015), the absence of nucleic acids in multiplex PCR indicates irreversible damage to the bacterial cells. The method used here appears to be more practical for producing axenic Nannochloropsis spp. ...
Highly nutritional microalgal species are extensively used in aquaculture as live feedstock. Due to difficulties in maintaining microalgae in axenic conditions, they represent a potential pathogen carrier and disease vector in aquaculture ponds. Photodynamic therapy (PDT) via singlet oxygen (¹O2) production is a promising sterilization technique in aquaculture. Here, we report on the sensitivity of aquaculture-relevant microalgae towards ¹O2 generated by the cationic photosensitizer TMPyP. Possible PDT sterilization protocols of contaminated microalgae cultures were evaluated using the luminescent bacterium Vibrio campbellii ISO7 as a model aquaculture pathogen. Species-specific sensitivity of microalgae to TMPyP-mediated PDT was demonstrated and found to be strongly influenced by the nature and architecture of their respective cell wall. While cytotoxicity was not evident against Nannochloropsis oculata, toxicity of ¹O2 was dose-, time- and light activation-dependent against Tisochrysis lutea, Tetraselmis chui, Chaetoceros muelleri and Picochlorum atomus. The ¹O2-resilient N. oculata was sterilized when incubated under light in the presence of V. campbellii ISO7 (up to 10⁷ CFU mL⁻¹) and 20 μM TMPyP; hence, TMPyP-based PDT sterilization of N. oculata could be suitable for aquaculture hatcheries. This study also suggests that PDT using cationic porphyrins such as TMPyP holds potential as an algicidal treatment in aquaria and aquaculture systems (but more research using opportunistic and toxic species is needed for confirmation).
... The ability of the bacteria to develop mutations that help their survival in the presence of antibiotics is responsible for the increasing number of resistant bacteria strains that will quickly become predominant in the microbial population [4][5][6][7][8]. The resistance development by the bacteria facing the conventional antibiotics have led the scientific community to increase efforts to find alternatives against to this emergent resistance [9][10][11][12]. ...
Photodynamic inactivation (PDI) is a therapeutic approach in study due to the ability to reduce or completely eliminate the bacterial strains without the development of resistance mechanisms. In this therapeutic methodology the cationic chlorins (Chls) with pyridinium or inverted pyridinium moieties are one of the photosensitizers exploited in our biological approaches. In this context, we synthesized and characterized new free-base and zinc(II) complexes of pyridinium or inverted pyridinium Chl derivatives (1b, 2, 2a and 2b, respectively) for the inactivation of Escherichia coli (E. coli). The PDI assay was performed with white light irradiation delivered at a fluence rate of 25 mW.cm-2. The obtained results of this study demonstrate high PDI efficiency of the zinc(II) metallated Chl 1b, reaching the detection limit of the bioluminescent method (5.2 log reduction) in 45 min of irradiation.
... The effect of the PS was evaluated based on the number of viable CFU per mL in comparison to a control that was not incubated with a PS (Figure 4). The dark toxicity effect of the synthesized 1-4 porphyrins was also compared to a commercially available tetracationic porphyrin derivative commonly used in PDI (6), and a neutral porphyrin derivative (5) [18,22,30,31]. The cationic porphyrin derivatives showed minimum dark toxicity to E. coli at concentrations of ≤1 µM (Figure 4). ...
Photodynamic inactivation (PDI) is a non-antibiotic option for the treatment of infectious diseases. Although Gram-positive bacteria have been shown to be highly susceptible to PDI, the inactivation of Gram-negative bacteria has been more challenging due to the impermeability properties of the outer membrane. In the present study, a series of photosensitizers which contain one to four positive charges (1–4) were used to evaluate the charge influence on the PDI of a Gram-negative bacteria, Escherichia coli (E. coli), and their interaction with the cell membrane. The dose-response PDI results confirm the relevance of the number of positive charges on the porphyrin molecule in the PDI of E. coli. The difference between the Hill coefficients of cationic porphyrins with 1–3 positive charges and the tetra-cationic porphyrin (4) revealed potential variations in their mechanism of inactivation. Fluorescent live-cell microscopy studies showed that cationic porphyrins with 1–3 positive charges bind to the cell membrane of E. coli, but are not internalized. On the contrary, the tetra-cationic porphyrin (4) permeates through the membrane of the cells. The contrast in the interaction of cationic porphyrins with E. coli confirmed that they followed different mechanisms of inactivation. This work helps to have a better understanding of the structure-activity relationship in the efficiency of the PDI process of cationic porphyrins against Gram-negative bacteria.
In this study, photostability and photodynamic antimicrobial performance of dye extracts from Hibiscus sabdariffa (HS) calyces, Sorghum bicolor (SB) leaf sheaths, Lawsonia inermis (LI) leaves and Curcuma longa (CL) roots were investigated in Acetate-HCl (AH) Buffer (pH 4.6), Tris Base-HCl (TBH) Buffer (pH 8.6), distilled water (dH2O), and Phosphate Buffer Saline (PBS, pH 7.2) using Bacillus subtilis as model for gram positive bacteria, Escherichia coli as model for gram negative bacteria, phage MS2 as model for non-envelope viruses and phage phi6 as model for envelope viruses including SARS CoV-2 which is the causative agent of COVID-19. Our results showed that the photostability of the dye extracts is in the decreasing order of LI > CL > SB > HS. The dye extract-HS is photostable in dH2O but bleaches in buffers—AH, TBH and PBS. The rate of bleaching is higher in AH compared to in TBH and PBS. The bleaching and buffers affected the photodynamic and non-photodynamic antimicrobial activity of the dye extracts. The photodynamic antibacterial activity of the dye extracts is in the decreasing order of CL > HS > LI > SB while the non-photodynamic antibacterial activity is in the decreasing order of LI > CL > HS > SB. The non-photodynamic antiviral activity pattern observed is the same as that of non-photodynamic antibacterial activity observed. However, the photodynamic antiviral activity of the dye extracts is in the decreasing order of CL > LI > HS > SB. Given their performance, the dye extracts maybe mostly suitable for environmental applications including fresh produce and food disinfection, sanitation of hands and contact surfaces where water can serve as diluent for the extracts and the microenvironment is free of salts.
The spread of antibiotic resistant pathogens and antibiotic resistance genes (ARGs) in the environment poses a serious threat to public health. However, existing methods are difficult to effectively remove antibiotic resistant pathogens and ARGs from the environment. In this study, we synthesized a new acridine-based photosensitizer, 2,7-dibromo-9-mesityl-10-methylacridinium perchlorate (YM-3), by the heavy atom effect, which could photodynamically inactivate antibiotic resistant pathogens and reduce ARGs by generating singlet oxygen (1O2) in an aqueous environment. The 1O2 yield of YM-3 was 4.9 times that of its modified precursor. YM-3 could reduce the culturable number and even the viable counts of methicillin-resistant Staphylococcus aureus and carbapenem-resistant Acinetobacter baumannii to 0 (inactivation rate > 99.99999%) after 2 and 8 h of low-intensity blue light (15 W/m2) irradiation, respectively. After 20 h of light exposure, the copy numbers of ARGs in both bacteria were reduced by 5.80 and 4.48 log, respectively, which might indicate that ARGs had been degraded. In addition, YM-3 still had an efficient bactericidal effect after five inactivation cycle. These characteristics of ultra-low light intensity requirement and efficient bactericidal ability make YM-3 have good application prospects for disinfection in indoor and sunlight environment.
Here, we investigated the bactericidal effects of two types of photoinduced reactive oxygen species (ROS), superoxide anion and singlet oxygen, on bacteria with distinct surface charges. We fabricated photofunctional polymer films (PFPFs) capable of generating both types of ROS, and they were subjected to photodynamic inactivation tests for 12 various strains of Acinetobacter baumannii. The results showed that the type I ROS (superoxide anion) was significantly dependent on the surface charge of the bacteria owing to charge-charge repulsion, while the type II ROS (singlet oxygen) was independent of the surface charge of the bacteria. These results would be significant in enhancing treatment efficiency in the clinical field.
Although the whole world is currently observing the global battle against COVID-19, it should not be underestimated that in the next 30 years, approximately 10 million people per year could be exposed to infections caused by multi-drug resistant bacteria. As new antibiotics come under pressure from unpredictable resistance patterns and relegation to last-line therapy, immediate action is needed to establish a radically different approach to countering resistant microorganisms. Among the most widely explored alternative methods for combating bacterial infections are metal complexes and nanoparticles, often in combination with light, but strategies using monoclonal antibodies and bacteriophages are increasingly gaining acceptance. Photodynamic inactivation (PDI) uses light and a dye termed a photosensitizer (PS) in the presence of oxygen to generate reactive oxygen species (ROS) in the field of illumination that eventually kill microorganisms. Over the past few years, hundreds of photomaterials have been investigated, seeking ideal strategies based either on single molecules (e.g., tetrapyrroles, metal complexes) or in combination with various delivery systems. The present work describes some of the most recent advances of PDI, focusing on the design of suitable photosensitizers, their formulations, and their potential to inactivate bacteria, viruses, and fungi. Particular attention is focused on the compounds and materials developed in our laboratories that are capable of killing in the exponential growth phase (up to seven logarithmic units) of bacteria without loss of efficacy or resistance, while being completely safe for human cells. Prospectively, PDI using these photomaterials could potentially cure infected wounds and oral infections caused by various multidrug-resistant bacteria. It is also possible to treat the surfaces of medical equipment with the materials described, in order to disinfect them with light, and reduce the risk of nosocomial infections.
The photoinactivation efficiency of antimicrobial photodynamic therapy (aPDT) with cationic porphyrin derivatives (CPDs) against multidrug-resistant (MDR) bacterial strain was assessed. MDR bacterial strains including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae were used. The CPDs named PM, PE, PN, and PL were synthesized as a photosensitizer (PS). A diode laser with a wavelength of 655 nm was used as a light source. Photoinactivation efficiency of the combinations formed with different energy density (50, 100, and 150 J/cm²) and PS concentrations (ranging from 3.125 µM and 600 µM) on each bacterial strain were evaluated. Toxicity of the aPDT combinations that showed a strong photoinactivation on the bacterial strains and dark toxicity of PSs and were evaluated on fibroblasts cells. In the aPDT experiments, survival reductions of up to 5.80 log₁₀ on E. coli, 5.90 log₁₀ on P. aeruginosa, 6.11 log₁₀ on K. pneumoniae and 6.78 log₁₀ on A. baumannii were obtained. There was an increase in the photoinactivation efficiency in parallel with increasing the energy density, and the best effect seen at an energy density of 150 J/cm2. PL did not show any toxic effect on fibroblasts. However, other PSs were toxic in fibroblasts at high concentrations. In this research, which reflected the results of in vitro experiments, aPDT provided potent photoinactivation against MDR clinical isolates. The research results lead to an in vivo wound model study of aPDT with CPD infected with an MDR clinical isolate.
Antimicrobial photodynamic therapy (aPDT) has gained prominence in microbiology, especially in treating non-invasive infections. Diseases such as mycobacteriosis, which causes localized infections and has a slow treatment, tend to be future targets for this type of technology. Therefore, this study aimed to explore, for the first time, the action of two isomeric Pd(II)-porphyrins on fast-growing mycobacterial strains (RGM). Tetra-cationic porphyrins (4-PdTPyP and 3-PdTPyP) were synthesized and applied against standard strains of Mycobacteroides abscessus subsp. abscessus (ATCC 19977), Mycolicibacterium fortuitum (ATCC 6841), Mycolicibacterium smegmatis (ATCC 700084), and Mycobacteroides abscessus subsp. massiliense (ATCC 48898). Reactive oxygen species (ROS) scavengers were used in an attempt to determine possible ROS produced by the photosensitizers (PS) under study. Moreover, the impact of porphyrin on the mycobacterial surface was further evaluated by atomic force microscopy (AFM), and we observed significant damage on cells walls and altered nanomechanical and electrostatic adhesion properties. The results presented herein show that the positively charged porphyrin at the meta position (3-PdTPyP) was the most efficient PS against the RGM strains, and its bactericidal activity was proven in two irradiation sessions, with singlet oxygen species being the main ROS involved in this process. This study demonstrated the therapeutic potential of porphyrins, especially the 3-PdTPyP derivative.
Bacterial infection has become an urgent health problem in the world. Especially, the evolving resistance of bacteria to antibiotics makes the issue more challenging, and thus new treatments to fight these infections are needed. Antibacterial photodynamic therapy (aPDT) is recognized as a novel and promising method to inactivate a wide range of bacteria with few possibilities to develop drug resistance. However, the photosensitizers (PSs) are not effective against Gram-negative bacteria in many cases. Herein, we use conjugated meso-tetra(4-carboxyphenyl)porphine (TCPP) and triaminoguanidinium chloride (TG) to construct self-assembled cationic TCPP-TG nanoparticles (NPs) for efficient bacterial inactivation under visible light illumination. The TCPP-TG NPs can rapidly adhere to both Gram-negative and Gram-positive bacteria and display promoted singlet oxygen (1O2) generation compared with TCPP under light irradiation. The high local positive charge density of TCPP-TG NPs facilitates the interaction between the NPs and bacteria. Consequently, the TCPP-TG NPs produce an elevated concentration of local 1O2 under light irradiation, resulting in an extraordinarily high antibacterial efficiency (99.9999% inactivation of the representative bacteria within 4 min). Furthermore, the TCPP-TG NPs show excellent water dispersity and stability during 4 months of storage. Therefore, the rationally designed TCPP-TG NPs are a promising antibacterial agent for effective aPDT.
Photocytotoxic effect induced by 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and 5,10,15,20-tetrakis[4-(3-N,N,N-trimethylaminepropoxy)phenyl]porphyrin (TAPP⁺⁴) was examined in Candida albicans to obtain information on the mechanism of photodynamic action and cell damage. For this purpose, the photokilling of the yeast was investigated under anoxic conditions and cell suspensions in D2O. Moreover, photoinactivation of C. albicans was evaluated in presence of reactive oxygen species scavengers, such as sodium azide and D-mannitol. The results indicated that singlet molecular oxygen was the main reactive species involved in cell damage. On the other hand, the binding and distribution of these porphyrins in the cells was observed by fluorescence microscopy. Morphological damage was studied by transmission electron microscopy (TEM), indicating modifications in the cell envelopment. Furthermore, deformed cells were observed after photoinactivation of C. albicans by toluidine blue staining. In addition, modifications in the cell envelope due to the photodynamic activity was found by scanning electron microscopy (SEM). Similar photodamage was observed with both porphyrin, which mainly produced alterations in the cell barriers that lead to the photoinactivation of C. albicans.
Mosquitoes are carriers of dangerous infectious disease pathogen all over the world. Through travel and global warming, tropical disease-carrying species such as Aedes, Anopheles and Culex spread beyond tropical and subtropical zones, even to Europe. Aim of this study is to investigate the potential of photodynamic agents to combat mosquito larvae. Three different photosensitizers were tested on Chaoborus spec. larvae: TMPyP and TPPS as antimicrobial photosensitizers and mTHPC, a PDT-drug against eukaryotic animal and human cells. Chaoborus spec. is a commercially available harmless species developing translucent larvae similar to the larvae of Aedes, Anopheles and Culex. The uptake of photosensitizers by the larvae was tested by fluorescence microscopy. All tested photosensitizers were observed in the intestinal tract of the living larvae, none of the photosensitizers was found in the larval tissue. In phototoxicity tests mTHPC and TPPS did not show any effect on the larvaewhile TMPyP killed the larvae efficiently. TPPS is an antimicrobial photosensitizer mainly phototoxic to Gram-positive bacteria. TMPyP is well known as an efficient photosensitizer against Gram-negative bacteria like most species of the intestinal flora. From this result, we conclude: the photodynamic Inactivation of intestinal flora leads to the death of mosquito larvae. The feasibility of mosquito larvae control by photodynamic inactivation of their intestinal flora instead of direct killing of the larvae is a promising alternative to other highly toxic insecticides. Compared to insecticides and other biochemical toxins, photosensitisers are not dark toxic. No resistances against photosensitizers are known so far. Thus, the dilution of the active substances by distribution in the environment, which promotes the formation of resistance in biocides of all kinds, does not pose a danger. Thus it reduces potential side effects on environment and human health.
The increasing world-wide rate of antibiotic resistance as well as the capacity of microorganisms to form biofilms, have led to a higher incidence of mortal infections that require alternative methods for their control. Antimicrobial photodynamic therapy (aPDT) emerged as an effective solution against resistant strains. The present work aims to evaluate the aPDT efficiency of a photosensitizer (PS) based on a low-cost formulation constituted by five cationic porphyrins (FORM) and its potentiation effect by KI on a broad spectrum of microorganisms under white light (380-700 nm, 25 W/m2). The aPDT assays were performed with different concentrations of FORM (0.1 to 5.0 μM) and 100 mM of KI on planktonic and biofilm forms of gram-positive (methicillin resistant Staphylococcus aureus-MRSA) and gram-negative (Escherichia coli resistant to chloramphenicol and ampicillin) bacteria, of the fungi Candida albicans and on a T4-like bacteriophage as a mammalian virus model. The results indicate that the FORM alone is an efficient PS to photoinactivate not only gram-negative and gram-positive bacteria, but also C. albicans, in planktonic and biofilm forms, and T4-like phage at low concentrations (<5.0 μM). The presence of KI enhanced the photodynamic effect of this FORM for all microorganisms on the planktonic form, allowing the reduction of PS concentration and treatment time. The results also show that the combination FORM/KI is highly efficient in the elimination of already well-established biofilms of E. coli,S. aureus and C. albicans. This effect is probably associated with longer-lived iodine reactive species produced during the aPDT treatment.
Photodynamic therapy (PDT) is based on the accumulation of a photosensitizer. such as a porphyrin or a chlorin, in a malignant tissue after its administration. Chlorins exhibit photophysical properties similar to those of the porphyrin macrocycles, but with intensified and red-shifted Q bands, making chlorin-containing systems even better candidates for PDT. In this contribution, we report the synthesis of 5,10,15-tris(pentafluorophenyl)-20-(4-pyridyl)porphyrin, (2) and its transformation to the novel chlorin derivatives 4. (5,10.20-tris(pentafluorophenyl)-15-(4-pyridyl)tetrahydro-1H-N-methyl-pyrrolo [3,4-b]porphyrin and 5, (5,10,15-tris(pentafluorophenyl)-20-(4pyridyl)-tetrahydro-1H-N-methyl-pyrrolo[3,4-b]porphyrin) by 1,3-dipolar cycloaddition with an azomethine ylide. The new products have been characterized by UV-Vis.H-1 NMR and FAB-MS. The photophysics, photochemical and photobleaching properties of chlorin 4 have been evaluated. Its quantum yield of photobleaching (phi(Pb), mol Einstein(-1)) was 0.047+/-0.014. In order to demonstrate the production of O-1(2) when 4 is used as a photosensitizer, uric acid tests have been carried out. The results indicate that chlorin 4 can be considered a promising photosensitizer in PDT.
Background and Objectives
Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach.ResultsOptimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a >4–5 log decrease in the microbial population after incubation times as short as 5–10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm2 and irradiation times shorter than 15 minutes.ConclusionsPDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections. Lasers Surg. Med. 38:468–481, 2006. © 2006 Wiley-Liss, Inc.
Antimicrobial photodynamic therapy (aPDT) has emerged in the clinical field as a potential alternative to antibiotics to treat microbial infections. No cases of microbial viability recovery or any resistance mechanisms against it are yet known. 5,10,15-tris(1-Methylpyridinium-4-yl)-20-(pentafluorophenyl)-porphyrin triiodide (Tri-Py(+)-Me-PF) was used as photosensitizer. Vibrio fischeri and recombinant Escherichia coli were the studied bacteria. To determine the bacterial recovery after treatment, Tri-Py(+)-Me-PF (5.0 microM) was added to bacterial suspensions and the samples were irradiated with white light (40 W m(-2)) for 270 minutes. Then, the samples were protected from light, aliquots collected at different intervals and the bioluminescence measured. To assess the development of resistance after treatment, bacterial suspensions were exposed to white light (25 minutes), in presence of 5.0 microM of Tri-Py(+)-Me-PF (99.99% of inactivation) and plated. After the first irradiation period, surviving colonies were collected from the plate and resuspended in PBS. Then, an identical protocol was used and repeated ten times for each bacterium. The results suggest that aPDT using Tri-Py(+)-Me-PF represents a promising approach to efficiently destroy bacteria since after a single treatment these microorganisms do not recover their viability and after ten generations of partially photosensitized cells neither of the bacteria develop resistance to the photodynamic process.
Owing to the increasing importance of aquaculture to compensate for the progressive worldwide reduction of natural fish and to the fact that several fish farming plants often suffer from heavy financial losses due to the development of infections caused by microbial pathogens, including multidrug resistant bacteria, more environmentally-friendly strategies to control fish infections are urgently needed to make the aquaculture industry more sustainable. The aim of this review is to briefly present the typical fish farming diseases and their threats and discuss the present state of chemotherapy to inactivate microorganisms in fish farming plants as well as to examine the new environmentally friendly approaches to control fish infection namely phage therapy and photodynamic antimicrobial therapy.
Photodynamic activity of chemical compounds towards microorganisms was first published at the turn of 20th century and it is based on the concept that a chemical compound, known as the photosensitizer, is localized preferentially in the microorganism and subsequently activated by low doses of visible light of an appropriate wavelength to generate reactive oxygen species that are toxic to the target microorganisms. Processes, in which absorption of light by a photosensitizer induces chemical changes in another molecule, are defined as photosensitizing reactions. Since the middle of the last century, antibacterial photosensitizing reactions were forgotten because of the discovery and the beginning of the Golden Age of antibiotics. Certainly, in the last decades the worldwide rise in antibiotic resistance has driven research to the development of new anti-microbial strategies. Different classes of molecules including phenothiazine, porphyrines, phthalocyanines, and fullerenes have demonstrated antimicrobial efficacy against a broad spectrum of antibiotic resistant microorganisms upon illumination. Due to their extended pi-conjugated system these molecules absorb visible light, have a high triplet quantum yield and can generate reactive oxygen species upon illumination. This mini-review will focus on some major advances regarding physical and chemical properties of photosensitizers and light sources that appear to be suitable in the field of antimicrobial photodynamic therapy. Currently, topical application of a photosensitizer on infected tissues and subsequent illumination seems to be the most promising feature of antimicrobial photodynamic therapy, thereby not harming the surrounding tissue or disturbing the residual bacteria-flora of the tissue.
In recent times photodynamic antimicrobial therapy has been used to efficiently destroy Gram (+) and Gram (-) bacteria using cationic porphyrins as photosensitizers. There is an increasing interest in this approach, namely in the search of photosensitizers with adequate structural features for an efficient photoinactivation process. In this study we propose to compare the efficiency of seven cationic porphyrins differing in meso-substituent groups, charge number and charge distribution, on the photodynamic inactivation of a Gram (+) bacterium (Enterococcus faecalis) and of a Gram (-) bacterium (Escherichia coli). The present study complements our previous work on the search for photosensitizers that might be considered good candidates for the photoinactivation of a large spectrum of environmental microorganisms.
Bacterial suspension (10(7) CFU mL(-1)) treated with different photosensitizers concentrations (0.5, 1.0 and 5.0 microM) were exposed to white light (40 W m(-2)) for a total light dose of 64.8 J cm(-2). The most effective photosensitizers against both bacterial strains were the Tri-Py+-Me-PF and Tri-Py+-Me-CO2Me at 5.0 microM with a light fluence of 64.8 J cm(-2), leading to > 7.0 log (> 99,999%) of photoinactivation. The tetracationic porphyrin also proved to be a good photosensitizer against both bacterial strains. Both di-cationic and the monocationic porphyrins were the least effective ones.
The number of positive charges, the charge distribution in the porphyrins' structure and the meso-substituent groups seem to have different effects on the photoinactivation of both bacteria. As the Tri-Py+-Me-PF porphyrin provides the highest log reduction using lower light doses, this photosensitizer can efficiently photoinactivate a large spectrum of environmental bacteria. The complete inactivation of both bacterial strains with low light fluence (40 W m(-2)) means that the photodynamic approach can be applied to wastewater treatment under natural light conditions which makes this technology cheap and feasible in terms of the light source.
Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.
A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
Those of us who have been developing the concept of a marine microbial food web have believed for many years that Protozoa are major movers of energy and nutrients. Indeed, Vernadskii (1926) repeatedly referred to the special significance of microorganisms, including specific references to Protozoa as movers of materials on a global, biogeochemical scale. However, until recently this remained more a matter of opinion than established fact. The conferees brought together an impressive body of facts regarding both the magnitude and the diversity of protozoan activity. That protozoa are a dominant force in the ocean’s metabolism is no longer in doubt. A comprehensive understanding of protozoan metabolism in the ocean is a prerequisite to a full understanding of and to quantitatively model cycles of carbon and other biologically essential elements on a global scale. During this conference we defined the limits of our knowledge of protozoan ecology and highlighted a number of pressing needs, both for basic understanding of the biosphere and its function and for answering questions of immediate practical importance.
There has been an explosion of research on marine microbial foodweb processes in the past decade. Today it is widely accepted that about 50% of the primary production in marine and fresh water is processed by bacteria each day (Williams, 1981; Cole et al., 1988). This striking finding was stimulated, as others have noted, by the introduction of convenient methods for the estimation of microbial biomass and activities in natural waters. Hobbie et al. (1977) and Watson et al. (1977) demonstrated conclusively that bacterial populations in the sea were large. By 1980, in addition to the pioneering and prescient work by Sorokin (e.g., Sorokin, 1971, 1973), reports of bacterial production measurements had begun to emerge (Sieburth et al., 1977; Karl, 1979; Larsson and Hagstrom, 1979; Fuhrman and Azam, 1980). Brock (1971) and Sieburth (1977) wrote early reviews on the subject, and Pomeroy (1974) introduced the importance of marine microbial processes to a large audience. In this chapter we review recent research on bacterial production in the ocean. The emphasis is on the open sea, but we will also discuss other marine habitats, partly because there are still few comprehensive studies of oceanic bacterial production. There is an equally large and rapidly growing literature on bacterial production in fresh waters (Cole et al., 1988; Currie, 1990) which deserves a review of its own, as well as comparison with the marine findings (Hobbie, 1988). We will not review related work in sediments, nor for the most part, related work on bacteriovores.
Photodynamic therapy is a platform technology which uses a combination of a photosensitizer, light and molecular oxygen to achieve selective destruction of a biological target. This methodology is already in use for the inactivation of microorganisms but its application in wastewater disinfection is incipient. In this work we tested the effect of three solid matrixes with immobilized porphyrins in the photoinactivation of a sewage bacteriophage. The phage inactivation to the limits of detection (reductions of about 7 log) with one of the tested materials, means that this insoluble material can be applied in wastewater disinfection with the same efficacy of the non-immobilized photosensitizer. The complete eradication of viruses with low light intensity means that this technology can be applied to wastewater disinfection under natural irradiation conditions during all year, including the cloudy days of winter. In addition, this is an inexpensive and easily applicable methodology.
In early summer, 1983, an acute disease characterized by ascites occurred among yellowtail fingerlings, Seriola quinqueradiata, cultured in a farm in the Seto Inland Sea, Japan, and accordingly virological investigations were carried out in July.
All three inoculants prepared from the viscera of the fish with ascites produced cytopathic effects (CPE) of pyknosis on RTG-2, CHSE-214 and EK-1 cells within 1.5-5 days after inoculation at 20°C. The virus replicated in CHSE-214 and EK-1 cells at temperature between 5-30°C. Electron micrographs revealed pentagonal or hexagonal particles of 62-69 nm. The virus was resistant to pH 3, pH 11, ether and chloroform and was stable at 56°C for 30 minutes. Replication of the virus was not inhibited by 5-Iodo-2'-deoxyuridine. Neutralization tests with antisera proved that this virus resembled to IPNV.
Pathogenicity of the virus was studied using yellowtail fingerlings (average body weight 1.1 g). Fish were exposed to the virus by immersion for 1 hour in seawater containing106.6 TCID50 /ml of the virus. The fish infected with the virus showed mortality of 62 % at water temperature of 20°C and 14 % at 25°C, while controls showed mortality of only 4 % at 20°C and 10% at 25°C, respectively. Moribund fish infected with the virus were often accompanied by severe ascites and hemorrhage in the liver. The virus reisolated from all the infected fish died.
The results strongly suggest that this virus was the causative agent of the ascites of yellowtail fingerlings. The agent was tentatively named yellowtail ascites virus (YAV).
The oxygen radical species responsible for the inactivation of phages by ascorbic acid was investigated, using four phages of different nucleic acid types. A scavenger for the superoxide anion radical (O2-) and scavengers for the hydroxyl radical (OH•) markedly prevented the inactivation of phages by ascorbic acid. Quenchers for singlet oxygen (1O2 slightly prevented the inactivation. Catalase fully prevented the inactivation, whereas superoxide dismutase only partially prevented it. Phages were markedly inactivated in an OH•- generating system, but not in an O2--generating system or an 1O2-generating system. These results, taken together, indicate that OH• is the major reactive species and that it is directly responsible for the inactivation of phages by ascorbic acid.
Unsaturated lipids in cell membranes, including phospholipids and cholesterol, are well-known targets of oxidative modification, which can be induced by a variety of stresses, including ultraviolet A (UVA)- and visible light-induced photodynamic stress. Photodynamic lipid peroxidation has been associated with pathological conditions such as skin phototoxicity and carcinogenesis, as well as therapeutic treatments such as antitumor photodynamic therapy (PDT). Lipid hydroperoxides (LOOHs), including cholesterol hydroperoxides (ChOOHs), are important non-radical intermediates of the peroxidative process which can (i) serve as in situ reporters of type I vs. type II chemistry; (ii) undergo one-electron or two-electron reductive turnover which determines whether peroxidative injury is respectively intensified or suppressed; and (iii) mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great. The purpose of this article is to review current understanding of photodynamic (UVA- or visible light-induced) lipid peroxidation with a special focus on LOOH generation and reactivity. Future goals in this area, many of which depend on continued development of state-of-the-art analytical techniques, will also be discussed.
Microorganisms have major roles in pond culture, particularly with respect to productivity, nutrient cycling, the nutrition of the cultured animals, water quality, disease control and environmental impact of the effluent. Management of the activities of microorganisms in food webs and nutrient cycling in ponds is necessary for optimising production, but the objectives will differ with the type of aquaculture, the species cultivated and the economics. Unlike the pastoralists who fertilise and seed pastures with selected species, fish farmers only fertilise ponds and hope that useful species of algae, bacteria and other microorganisms will develop. Factors controlling the composition and development of blooms of both desired and undesirable species of algae and bacteria need to be investigated. Analysis of the complex food webs in ponds, combined with measurements of primary productivity, C cycling through bacteria, Zooplankton and meiofaunal biomass changes and nitrogen cycling will provide the basic data for generating a model to describe and predict fish productivity in ponds. Algae and bacteria have often been regarded as single groups of organisms by aquatic biologists, but there is a great diversity of species of each, with different roles and interactions in their ecosystems. This fact, together with the many environmental variables, makes it difficult to develop a single comprehensive, predictive model as a tool for managing food webs and water quality in ponds, but small models specific to given pond systems or stages of grow-out are likely to be successful.
Meta(tetrahydroxyphenyl)chlorin (m-THPC) is a new photosensitizer currently undergoing clin. trials at Lausanne's CHUV hospital for photodynamic therapy (PDT) of early cancer in the upper aerodigestive tract. The illumination of m-THPC with light at 650 nm in aq. soln. contg. 10% fetal calf serum (FCS) causes two simultaneously occurring processes: its photodegrdn. and the formation of a more stable photoproduct absorbing at 320 nm. The photodegrdn. quantum yield (FPb) of m-THPC is found to be of the order of 1.5*10-5 in 10% FCS. A strong dependence on oxygen concn. of the photodegrdn. and the formation of photoproducts has been obsd. Indeed, the m-THPC presents rather low FPb under N2-satd. conditions: 6.9*10-6. In aerobic conditions, the photodegrdn. as well as the formation of photoproducts, have been competitively inhibited by known singlet oxygen (1O2) quenchers. The addn. of superoxide dismutase (SOD), catalase or desferal, known quenching agents of type I mechanisms, has little or no effect on the rate of photobleaching and photoproduct formation of m-THPC. m-THPC generates 1O2 with a quantum yield of 0.3 in ethanol soln. as detd. by photo-oxidn. expts. using 1,3-diphenylisobenzofuran (DPBF) as substrate. The rate and quantum yield of DPBF photo-oxidn. are found to increase with increasing substrate concn. and decrease in phosphate buffer soln. (FD=0.01), due to the partially hydrophilic character of m-THPC. In addn., the reaction of 1O2 with TEMP (2,2,6,6-tetramethyl-4-piperidone) in combination with ESR (EPR) detection has been used to det. the formation of 1O2 by m-THPC in ethanol soln.
The community structure of attached and free-living bacteria in the Aegean Sea (eastern Mediterranean Sea) was analyzed with use of terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting. Since the Aegean Sea is characterized by rather small temperature fluctuations between surface and deep-water layers, it represents an ideal study site to determine the variations in the community structure of bacteria with depth, since environmental factors other than temperature are likely to determine depth zonation of bacteria. The analysis of 132 T-RFLP electropherograms indicated pronounced differences among the attached and free-living bacterial communities de- fined as operational taxonomic units (OTUs). Distinct vertical differences of attached and free-living OTUs were found between mesopelagic waters (200 m depth) and the upper mixed water column (10-200 m). Attached and free-living OTUs differed considerably throughout the water column, with only 35% for the South Aegean and 24% for the North Aegean of all OTUs in both free-living and attached OTUs. Approximately 50% of attached and free-living OTUs were present throughout the water column. Fingerprinting analysis using 16S rRNA indicated that only 14% of the attached and 33% of the free-living OTUs were identical to the 16S rDNA fingerprints. The distribution of free-living versus attached bacteria as obtained in this study suggests that even in the absence of temperature as a major selective factor, a distinct deep-water bacterial community exists (particularly in the free-living mode). The deep-water free-living bacterial community appears to be as compositionally complex as the surface water free-living bacterial community.
For the fist time worldwide we report high amplitude NIR-Luminescence signals, measured in pig skin. The measurements are achieved with a recently developed setup that was shown to provide superior performance for 1O2- luminescence detection in vitro. This setup has been adapted to allow now the detection of singlet oxygen signals in vivo. Pig ears were used for first measurements as a widely accepted in vivo model for human skin.
The upsurge in multidrug-resistant bacteria, e.g., methicillin-resistant Staphylococcus aureus (MRSA), in the healthcare milieu represents a serious clinical problem with associated high morbidity and mortality. Since resistant strains are selected by the overuse of single-site/mode-of-action therapeutics, different approaches to bacterial eradication are required. Similarly, traditional disinfection protocols for donated blood and blood products are inadequate in the face of the AIDS pandemic and other emerging pathogens. Photosensitizing drugs operate via the generation of reactive oxygen species upon illumination in situ. The lack of microbial resistance mechanisms, e.g., against singlet oxygen, ensures a wide range of topical/local applications across bacteria, viruses, yeasts and protozoa. This underlines the potential of photoantimicrobial chemotherapy (PACT) in infection control, whether in antisepsis or in the inhibition of transfusion/transmission of disease.
A comparison was made of membrane protein patterns of various Aeromonas salmonicida strains, initially isolated from different habitats with respect to fish species affected, pathological entity, and geographic location of the outbreak of the disease. A major protein with a molecular weight of 54 000 was found in all autoagglutinating strains, whereas this protein is present in low amounts, or not at all, in non-autoagglutinating strains. Evidence for a causal relationship between the presence of this protein and the phenomenon of autoagglutination came from the observation that a change of the growth medium led simultaneously to an almost complete loss of the additional cell envelope protein and the property of autoagglutination. As it has already been reported that autoagglutination is correlated with the presence of an additional cell surface layer, we hypothesize that the additional cell envelope protein is the (major) subunit of this layer. The application of the gel immuno radio assay, an immunological technique suited to detect antigens in a gel, revealed that the additional cell envelope proteins of all tested strains are immunologically related. The possibility to the use of this protein as a component of a vaccine against A. salmonicida infections is discussed.
Spatial and temporal patterns in the flux of sinking organic matter are central to the understanding of elemental dynamics and food-web energetics in the global ocean1-3. Heterotrophic bacteria have been shown to play a part in the decomposition of large, rapidly sinking organic particles within and below the euphotic zone4-8. These previous studies suggest that decomposition by attached bacteria can explain only a trivial fraction of the observed decrease in the flux of organic matter with increasing depth. We report here that free-living bacteria, rather than the particle-feeding zooplankton, are the principal mediators of particle decomposition in the central north Pacific gyre and the eutrophic Santa Monica basin. We suggest that bacterial growth in the mesopelagial gives rise to the large-scale production of fine (0.3-0.6 mum), non-sinking particles at the expense of large, rapidly sinking particles. Our results have implications for models of biogeochemical dynamics of organic particles and surface-reactive materials such as radionu-clides in the ocean's interior3,9.
This work deals with a new approach of grafting cellulose surface fibres by polycaprolactone macromolecular chains in heterogeneous conditions via click-chemistry. Thus, cellulose esters were prepared by reacting Avicel with undecynoic acid, in order to prepare cellulose substrate bearing multiple CC-terminated hairs. The prepared modified Avicel substrates were characterised by FTIR, XPS spectroscopy, elemental analyses and showed that the grafting have indeed occurred. The degree of substitution of the prepared cellulose esters was around 0.1. In parallel, polycaprolactone-diol (PCL) was converted to azido-derivative and the ensuing products characterised by FTIR and 13C-NMR spectroscopy. Both methods confirmed the success of such modification. Finally, cellulose esters were reacted with azido-PCL grafts in heterogeneous conditions through “click chemistry”. The thus prepared modified cellulose substrates were characterized by FTIR and XPS spectroscopy as well as elemental analyses. The three techniques confirmed the occurrence of the grafting. A weight gain of 20% was achieved.
The prevalence of antibiotic resistant bacteria necessitates exploration of alternative approaches to treat hospital and community acquired infections. The aim of this study was to determine whether bacterial pathogens develop resistance to antimicrobial photodynamic therapy (aPDT) during repeated sub-lethal challenge. Antibiotic sensitive and resistant strains of S. aureus and antibiotic sensitive E. coli were subjected to repeat PDT treatments using a methylene blue photosensitizer formulation and 670 nm illumination from a non-thermal diode laser. Parameters were adjusted such that kills were
The worldwide rise in antibiotic resistance by microorganisms has stimulated intensive research toward the development of alternative therapeutic strategies. Photodynamic therapy (PDT) is emerging as a promising modality for the treatment of localized microbial infections. Studies on the relationship between the chemical structure of photosensitising agents and their phototoxicity against microbial pathogens led to the identification of a selected number of compounds with optimal cytocidal effects. These include phenothiazine, porphyrin and phthalocyanine derivatives, whose molecule has been engineered to introduce the following features: (a) presence of cationic moieties, preferably due to quaternarized amino groups; (b) introduction of at least one N-alkyl group having a relatively long hydrocarbon chain; (c) overall amphiphilic character to promote the partitioning in the plasma membrane. Studies on cell cultures indicate that PDT is endowed with favourable properties to act as an antimicrobial modality: (a) broad spectrum of action, since one irradiation protocol can be used to obtain the inactivation of different groups of pathogens, such as Gram-positive and Gram-negative bacteria, yeasts, mycoplasmas and protozoa in both vegetative and cystic stages; (b) fast association with microbial cells, which allows irradiations to be performed after incubation times as short as 5-10 min., thereby guaranteeing a high selectivity as compared with host tissues; (d) high photoinactivation efficiency, since a 5-6 log decrease in microbial population is obtained by irradiation under mild conditions; (e) photosensitising activity independent of the antibiotic-resistance spectrum of the given pathogen; (f) lack of selection of photoresistant strains upon repeated treatment and minimal risk to induce the onset of mutagenic processes. Initial clinical trials involve the treatment of chronic ulcers and selected oral infections.
Fifteen amino acids were evaluated for antioxidant activity in a linoleate emulsion oxidized by hemoglobin. Cysteine was the only amino acid with significant antioxidant activity, which was as great as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), or e-tocopherol. Ten proteins containing free or latent sulfhydryl groups were evaluated for antioxidant activity in both native and reduced states. Native proteins, containing few or no measurable sulfhydryl groups, had little or no antioxidant activity. Proteins treated with NaBH4 had greatly increased sulfhydryl groups and antioxidant activity. Treatment of reduced proteins or cysteine with iodoacetic acid (IAA) eliminated both sulfhydryl groups and antioxidant activity. Cysteine and reduced bovine serum albumin had optimum antioxidant activity at pH 8.6.
The mechanism ofStaphylococcus aureus inactivation by deuteroporphyrin (DT) and light was studied with singlet oxygen quenchers or hydroxyl radical scavengers. The light-activated DT (10 /ml) reduced the viability of the culture to less than 1%, whereas methionine, tryptophan, and 1,4-diazabicyclo-2,2,2-octane (DBCO) used as singlet oxygen quenchers provided almost 60% protection. Propylgallate, which is a hydroxyl free radical scavenger, also provided 60% protection. The presence of a singlet oxygen quencher and propylgallate provided almost complete protection from inactivation (96%). Photoinactivation in the absence of culture media (in saline) increased the killing rate and decreased the ability of the singlet oxygen quenchers to protect. In the same conditions damage from hydroxl free radicals was well protected by propyl gallate. The present results indicate thatS. aureus photoinactivation by DT and light is mediated by both singlet oxygen and hydroxyl free radicals.
New hydrophilic immobilized photosensitizers (heterogeneous phase) were synthesized that overcome some disadvantages of the use of homogeneous phase sensitizers for detoxification and disinfection of water. The chosen sensitizers, based on porphyrin moieties, were bound on poly(methyl methacrylate) (PMMA). The measured production rate of singlet oxygen is significantly higher than that of the well-known rose bengal immobilized on Merrifield polymer. The sensitive polymer can be used for detoxification and disinfection of polluted water.
Using Escherichia coli and Enterococcus faecalis as model microorganisms, water disinfection has been demonstrated with singlet molecular oxygen (a reactive oxygen species) photogenerated by polymer-supported Ru(II) sensitizers and solar light. Both laboratory and sunlight tests were performed. The Ru(II) polypyridyl complex and its insoluble support have been optimized to provide maximum efficiency of singlet oxygen production and contact with the microorganism.
A meso-substituted cationic porphyrin (TMPyP) showed a photocytotoxicity against Gram-positive and Gram-negative bacteria. In order to determine the mechanism involved in the phototoxicity of this photosensitizer, electron paramagnetic resonance (EPR) experiments with 2,2,6,6-tetramethyl-4-piperidone (TEMP), a specific probe for singlet oxygen, and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were carried out with illuminated TMPyP. An EPR signal characteristic of TEMP-singlet oxygen (TEMPO) adduct formation was observed, which could be ascribed to singlet oxygen (1O2) generated by TMPyP photosensitization. The signal for the DMPO spin adduct of superoxide anion (DMPO-OOH) was observed in DMSO solution but not in aqueous conditions. However, an EPR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO-OH) was observed in aqueous conditions. The obtained results testify a primary hydroxyl radical (OH) generation probably from superoxide anion via the Fenton reaction and/or via Haber-Weiss reaction. Gram-positive and Gram-negative bacteria inactivation by TMPyP photosensitization predominantly involved Type II reactions mediated by the formation of 1O2, as demonstrated by the effect of quenchers for 1O2 and scavengers for OH (sodium azide, thiourea, and dimethylsulphoxide). Participation of other active oxygen species cannot however be neglected since Type I reactions also had a significant effect, particularly for Gram-negative bacteria. For Gram-negative bacteria the photoinactivation rate was lower in the presence of superoxide dismutase, a specific scavenger, and/or catalase, an enzyme which specifically eliminates H2O2, but was unchanged for Gram-positive bacteria. The generation of 1O2, and OH by TMPyP photosensitization indicated that TMPyP maintained a photodynamic activity in terms of Type I and Type II mechanisms.
Airborne micro-organisms are ubiquitous in the atmosphere where they can remain alive and be transported over long distances, thus colonizing new environments. Despite their great importance in relation to ecological and socio-economical issues (bio-terrorism, health, etc.) very few studies have been carried out in this field.In this study, the structure of the microbial community present in atmospheric water samples from clouds at the Puy de Dôme (alt 1465 m, Massif Central, France) is described and the metabolic potential of some bacteria is investigated. The total microflora has been quantified by epifluorescence microscopy, while the cultivable aerobic micro-organisms were isolated. Bacteria were identified by 16S DNA sequencing and fungi by morphological criteria. The total bacterial count reached about 3×104 cells m−3 of cloud volume (1×105 cells mL−1 of cloud water), of which less than 1% are cultivable. Most of the isolated micro-organisms, including 12 fungal and 17 bacterial strains, are described here for the first time in atmospheric water. Many bacterial strains seem to be adapted to the extreme conditions found in cloud water (pH, T°, UV radiations, etc.). Comparison of the two samples (March 2003) shows that pH can be a major factor controlling the structure of this community: an acidic pH (Sample 1: pH=4, 9) favours the presence of fungi and spore-forming bacteria, while a more neutral pH (Sample 2: pH=5, 8) favours greater biodiversity. We have also shown, using in situ 1H NMR, that most of the isolated bacteria are able to degrade various organic substrates such as formate, acetate, lactate, methanol and formaldehyde which represent the major organic compounds present in cloud water. In addition, the detection of intermediates indicated preferential metabolic routes for some of the strains.
The aim of this review is to compile some dispersed literature published about different aspects of the most threatening bacterial diseases occurring in fish cultured in marine waters worldwide such as vibriosis, “winter ulcer”, photobacteriosis, furunculosis, flexibacteriosis, “winter disease”, streptococcosis, lactococcosis, BKD, mycobacteriosis and piscirickettsiosis. Therefore, the geographic distribution of each disease and the main host species affected, together with the biochemical and antigenic diversity existing in the aetiologic agents are described. In addition, the genetic studies that have been performed to determine the possible existence of intraspecific heterogeneity or clonal lineages within each pathogen are included. We review also in brief the classical methods to isolate the microorganisms from their hosts as well as the serological and/or genetic tools for a rapid diagnosis of the diseases. Finally, the current status in the development of vaccination strategies to prevent these bacterial diseases is also addressed.
Exposure to visible-light causes the photoinactivation of certain bacteria by a process that is believed to involve the photo-stimulation of endogenous intracellular porphyrins. Studies with some bacterial species have reported that this process is oxygen-dependent. This study examines the role of oxygen in the visible-light inactivation of Staphylococcus aureus. Suspensions of S. aureus were exposed to broadband visible-light under both oxygen depletion and oxygen enhancement conditions to determine whether these environmental modifications had any effect on the staphylococcal inactivation rate. Oxygen enhancement was achieved by flowing oxygen over the surface of the bacterial sample during light inactivation and results demonstrated an increased rate of staphylococcal inactivation, with approximately 3.5 times less specific dose being required for inactivation compared to that for a non-enhanced control. Oxygen depletion, achieved through the addition of oxygen scavengers to the S. aureus suspension, further demonstrated the essential role of oxygen in the light inactivation process, with significantly reduced staphylococcal inactivation being observed in the presence of oxygen scavengers. The results of the present study demonstrate that the presence of oxygen is important for the visible-light inactivation of S. aureus, thus providing supporting evidence that the nature of the mechanism occurring within the visible-light-exposed staphylococci is photodynamic inactivation through the photo-excitation of intracellular porphyrins.
Carbohydrate-decorated meso-tetraarylporphyrins P-G and P-C were synthesized via Lewis-acid catalyzed condensation of acetylated carbohydrate-substituted benzaldehydes and pyrrole. Their efficiency of singlet oxygen production was compared with the corresponding non-substituted porphyrin. The oxidation of the spin trap molecule TEMP (2,2,6,6-tetramethyl-4-piperidone) by singlet oxygen to TEMPO was measured by ESR spectroscopy, showing higher reaction rates for the sugar porphyrins. These results were corroborate by laser flash photolysis measurements that resulted in higher triplet lifetimes of glucosyl- and cellobiosyl porphyrins in comparison with tetrakis(4-hydroxyphenyl)porphyrin. Low ee was detected in the photooxygenation of ethyl tiglate.
We appreciate the comments of Drs. Kaye and Hill on our article. We are pleased that these investigators concurred with our impression that photodynamic therapy (PDT) may potentially be useful in the treatment of posterior fossa tumors. Several interesting questions were raised about the technical aspects of our method of determining tissue Photofrin levels in tumor and surrounding normal brain. Our laboratory concurs with their feelings about the limitations of relative fluorescence. We have therefore conducted additional investigations with radiolabeled Photofrin to quantify tissue levels by yet another technique.
Construction of 16S rDNA libraries was undertaken to examine the structure of free-living bacterial communities in the estuarine system Ria de Aveiro, Portugal. Samples were collected in April 2002, from two sites representing marine (station N1) and brackish (station I6) water zones. Clones were characterized by RFLP patterns and sequence analysis of representative clones revealed that both libraries were clearly dominated by alpha-proteobacteria, followed by gamma-proteobacteria and beta-proteobacteria. Clones affiliated with the delta-proteobacteria, Verrucomicrobia, Bacteroidetes and Actinobacteria were exclusive of station I6 and sequences related to the Firmicutes were only found in station N1. Sequences retrieved are included in only a few major bacterial divisions and in general, shared a high degree of homology with sequences deposited in nucleotide databases, and recovered from aquatic environments of diverse geographic regions. Differences between the two sites may reflect adaptation to different environmental conditions, especially salinity. The pattern of prokaryotic diversity is comparable to other coastal and estuarine environments previously studied.
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.
The study of PDI of Bacillus cereus endospores, taken as model-endospores, using porphyrin derivatives differing in the number of positive charges and in the meso-substituent groups, showed that neutral, monocationic and dicationic porphyrins are quite ineffective, in contrast with the tri- and tetra-cationic molecules. The most effective porphyrin is a tricationic porphyrin with a meso-pentafluorophenyl group. With this photosensitizer (PS), at 0.5 micromol l(-1), a reduction of 3.5 log units occurs after only 4 min of irradiation. None of the porphyrin derivatives showed toxicity in the absence of light.
Some porphyrin derivatives are efficient PSs for the inactivation of bacterial endospores and should be considered in further studies. Small modifications in the substituent groups, in addition to charge, significantly improve the effectiveness of the molecule as a PS for endospore inactivation.
Tetrapyrrolic macrocycles should be regarded as worthy to explore for the PDI of spore-producing gram-positive bacteria. The development of molecules, more selective and effective, emerges as a new objective.
Methylene blue (MB) has been shown to photoinduce the direct inactivation of Gram-positive bacteria Staphylococcus aureus (S. aureus) in water. We have investigated the mechanism of S. aureus photoinactivation conducting firstly sodium azide (reactive ion N3−), as a good physical singlet oxygen quencher, then the amino acids tryptophan (Trp), as a non-specific singlet oxygen quencher and the mannitol, as an hydroxyl free radical scavenger. Inactivation of MB photosensitization is the antioxidants type dependent. When the bacteria was treated with MB (20 μM) under light during 10 min of exposure, it was found that survival fraction had decreased dramatically to about 31.27±5.39%. The presence of sodium azide and Trp failed to shown any protection from the MB photodynamic activity. In the presence of mannitol, S. aureus could be protected, reaching a protection level of about 27%. It is possible that the photodynamic activity of MB occurred in part, via a Type I mechanism in which •OH was produced.
The interactions between MB and S. aureus were studied spectrophotometrically. This demonstrated that a metachromatic reaction took place between MB and S. aureus bacteria. Furthermore, S. aureus bacteria induced additional dimerization of MB.
A faster and simpler method to monitor the photoinactivation process of Escherichia coli involving the use of recombinant bioluminescent bacteria is described here. Escherichia coli cells were transformed with luxCDABE genes from the marine bioluminescent bacterium Vibrio fischeri and the recombinant bioluminescent indicator strain was used to assess, in real time, the effect of three cationic meso-substituted porphyrin derivatives on their metabolic activity, under artificial (40 W m(-2)) and solar irradiation (approximately 620 W m(-2)). The photoinactivation of bioluminescent E. coli is effective (>4 log bioluminescence decrease) with the three porphyrins used, the tricationic porphyrin Tri-Py+-Me-PF being the most efficient compound. The photoinactivation process is efficient both with solar and artificial light, for the three porphyrins tested. The results show that bioluminescence analysis is an efficient and sensitive approach being, in addition, more affordable, faster, cheaper and much less laborious than conventional methods. This approach can be used as a screening method for bacterial photoinactivation studies in vitro and also for the monitoring of the efficiency of novel photosensitizer molecules. As far as we know, this is the first study involving the use of bioluminescent bacteria to monitor the antibacterial activity of porphyrins under environmental conditions.
Disease problems constitute the largest single cause of economic losses in aquaculture. In 1988, channel catfish producers lost over 100 million fish worth nearly $11 million. Estimates for 1989 predict even higher losses. The trout industry reported 1988 losses of over 20 million fish worth over $2.5 million. No data are available on losses sustained by producers of shellfish. Bacterial infections constitute the most important source of disease problems in all the various types of production. Gram-negative bacteria cause epizootics in nearly all cultured species. Fungal diseases constitute the second most important source of losses, especially in the culture of crustaceans and salmon. External protozoan parasites are responsible for the loss of large numbers of fry and fingerling fin fishes and are a cause of epizootics among young shellfish. The number of therapeutants approved by the Food and Drug Administration is limited. Research to support the registration of promising therapeutic agents is urgently needed.