Effectiveness Of Photodynamic Inactivation Research Articles

Morphological aspects and the effectiveness of photodynamic inactivation against Rhizopus oryzae in different life cycles.

Mucormycosis is an extremely aggressive fungal disease with a high mortality rate, especially in people with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae. The treatments used are based on high doses of antifungals, associated with surgical resections, when it is possible. However, even with this aggressive treatment, the estimated attributable mortality rate is high. There is therefore a need to develop adjuvant treatments. Photodynamic Inactivation (PDI) may be an auxiliary therapeutic option for mucormycosis. Due to the lack of reports in the literature on the morphology and photodynamic inactivation of R. oryzae, characterization of the fungus using Confocal Microscopy and Transmission Electron Microscopy, and different protocols using Photodithazine® (PDZ), a chlorin e6 compound, as a photosensitizer, were performed. The fungus growth rate under different concentrations and incubation times of the photosensitizer and its association with the surfactant Sodium Dodecyl Sulphate (SDS) was evaluated. For the hyphae, both in the light and dark phases, in the protocols using only PDZ, no effective photodynamic response was observed. Meanwhile with the combination of SDS 0.05% and PDZ, inhibition growth rates of 98% and 72% were achieved for the white and black phase, respectively. In the conidia phase, only a 1.7 log10 reduction of the infective spores was observed. High concentration of melanin and the complex and resistant structures, especially at the black phase, results in a high limitation of the PDI inactivation response. The combined use of the SDS resulted in an improved response, when compared to the one obtained with the amphotericin B treatment.

Wastewater Disinfection with Photodynamic Treatment and Evaluation of its Ecotoxicological Effects

Research has demonstrated the presence of viruses in wastewater (WW), which can remain viable for a long period, posing potential health risks. Conventional WW treatment methods involving UV light, chlorine and ozone efficiently reduce microbial concentrations, however, they produce hazardous byproducts and microbial resistance that are detrimental to human health and the ecosystem. Hence, there is a need for novel disinfection techniques. Antimicrobial Photodynamic Inactivation (PDI) emerges as a promising strategy, utilizing photosensitizers (PS), light, and dioxygen to inactivate viruses. This study aims to assess the efficacy of PDI by testing Methylene Blue (MB) and the cationic porphyrin TMPyP as PSs, along a low energy consuming white light source (LED) at an irradiance of 50 mW/cm2, for the inactivation of bacteriophage Phi6. Phi6 serves as an enveloped RNA-viruses surrogate model in WW. PDI experiments were conducted in a buffer solution (PBS) and real WW matrices (filtered and non-filtered). Considering the environmental release of the treated effluents, this research also evaluated the ecotoxicity of the resulting solution (post-PDI treatment effluent) on the model organism Daphnia magna, following the OECD immobilization technical 202 guideline. Daphnids were exposed to WW containing the tested PS at different concentrations and dilutions (accounting for the dilution factor during WW release into receiving waters) over 48 h. The results indicate that PDI with MB efficiently inactivated the model virus in the different aqueous matrices, achieving reductions superior to 8 log10 PFU/mL, after treatments of 5 min in PBS and of ca. 90 min in WW. Daphnids survival increased when subjected to the PDI-treated WW with MB, considering the dilution factor. Overall, the effectiveness of PDI in eliminating viruses in WW, the fading of the toxic effects on daphnids after MB’ irradiation and the rapid dilution effect upon WW release in the environment highlight the possibility of using MB in WW PDI-disinfection.

Methylene blue@silver nanoprisms conjugates as a strategy against Candida albicans isolated from balanoposthitis using photodynamic inactivation

Balanoposthitis can affect men in immunocompromised situations, such as HIV infection and diabetes. The main associated microorganism is Candida albicans, which can cause local lesions, such as the development of skin cracks associated with itching. As an alternative to conventional treatment, there is a growing interest in the photodynamic inactivation (PDI). It has been shown that the association of photosensitizers with metallic nanoparticles may improve the effectiveness of PDI via plasmonic effect. We have recently shown that the association of methylene blue (MB), a very known photosensitizer, with silver prismatic nanoplatelets (AgNPrs) improved PDI of a resistant strain of Staphylococcus aureus. To further investigate the experimental conditions involved in PDI improvement, in the present study, we studied the effect of MB concentration associated with AgNPrs exploring spectral analysis, zeta potential measurements, and biological assays, testing the conjugated system against C. albicans isolated from a resistant strain of balanoposthitis. The AgNPrs were synthesized through silver anisotropic seed growth induced by the anionic stabilizing agent poly(sodium 4-styrenesulfonate) and showed a plasmon band fully overlapping the MB absorption band. MB and AgNPrs were conjugated through electrostatic association and three different MB concentrations were tested in the nanosystems. Inactivation using red LED light (660 nm) showed a dose dependency in respect to the MB concentration in the conjugates. Using the highest MB concentration (100 µmol⋅L−1) with AgNPr, it was possible to completely inactivate the microorganisms upon a 2 min irradiation exposure. Analyzing optical changes in the conjugates we suggest that these results indicate that AgNPrs are enhancers of MB photodynamic action probably by a combined mechanism of plasmonic effect and reduction of MB dimerization. Therefore, MBAgNPrs can be considered a suitable choice to be applied in PDI of resistant microorganisms.

Application of photodynamic inactivation against pathogens of urinary tract infections

Photodynamic inactivation (PDI) is an alternative to antibiotic therapy method for biocidal action against microorganisms, which can be used for lithotripsy and sanitation of the bladder cavities. Objective. Selection of parameters and application PDI against uropathogenic microorganisms. Materials and Methods. In this study we used bacterial strains isolated from urine samples of patients. Differentiation media and biochemical plates were used for identification of microorganisms. The sensitivity of uropathogenic microorganisms to PDI was studied on pure cultures and in native urine. The photosensitizer “Photoditazine” (50 µg/ml) was used in the work, as well as Triton X-100 (5 % vol.) was applying to increase the permeability of the cell wall of gram-negative microorganisms. The samples were irradiated by a medical laser device “Latus-K” with a wavelength of 662 nm. To assess the effectiveness of PDI, the values of the logarithmic decrease of colony-forming unit (CFU) of the microorganisms were calculated. Statistical analysis was made by Statistica 10.0 and Mann-Whitney criterion. Results. 50 strains of uropathogens belonging to 18 species were isolated from 36 samples of native urine. Among them, the most common were S. aureus, E. coli, P. aeruginosa, K. pneumoniae. The value of logarithmic decrease in CFU for gram-positive bacteria ranged from 5 to 6, which corresponds to inactivation 99.999-99.9999% of bacterial cells in a sample. For gram-negative strains, this value was slightly lower and ranged from 4 to 5.5, which, nevertheless, corresponds to inactivation 99.99-99.999% of CFU bacteria. The addition of Triton X-100 increase the efficiency from 46% to 99.99% for E. coli, from 99% to 99.99% for P. mirabilis, from 16% to 94% for K. pneumoniae and from 97% to 99.999% for P. aeruginosa. It should be noted that the PDI was affect microorganisms both in isolated pure cultures and in native urine. Conclusions. Photodynamic inactivation may be considered as an alternative to antibiotic therapy method of biocidal action against uropathogenic microorganisms.

Photodynamic Inactivation Using Natural Bioactive Compound Prevents and Disrupts the Biofilm Produced by Staphylococcus saprophyticus.

Staphylococcus saprophyticus, the food-borne bacteria present in dairy products, ready-to-eat food and environmental sources, has been reported with antibiotic resistance, raising concerns about food microbial safety. The antimicrobial resistance of S. saprophyticus requires the development of new strategies. Light- and photosensitizer-based antimicrobial photodynamic inactivation (PDI) is a promising approach to control microbial contamination, whereas there is limited information regarding the effectiveness of PDI on S. saprophyticus biofilm control. In this study, PDI mediated by natural bioactive compound (curcumin) associated with LED was evaluated for its potential to prevent and disrupt S. saprophyticus biofilms. Biofilms were treated with curcumin (50, 100, 200 µM) and LED fluence (4.32 J/cm2, 8.64 J/cm2, 17.28 J/cm2). Control groups included samples treated only with curcumin or light, and samples received neither curcumin nor light. The action was examined on biofilm mass, viability, cellular metabolic activity and cytoplasmic membrane integrity. PDI using curcumin associated with LED exhibited significant antibiofilm activities, inducing biofilm prevention and removal, metabolic inactivation, intracellular membrane damage and cell death. Likewise, scanning electronic microscopy observations demonstrated obvious structural injury and morphological alteration of S. saprophyticus biofilm after PDI application. In conclusion, curcumin is an effective photosensitizer for the photodynamic control of S. saprophyticus biofilm.

Nanotechnology in antimicrobial photodynamic inactivation

Photodynamic therapy (PDT) and photodynamic inactivation (PDI) are technologies that utilize visible light and photosensitizers (PS) to inactivate cells. PDT is currently in use for the treatment of several types of tumors. Although cancer has been successfully treated with PS and light, antimicrobial PDI is emerging as a new treatment modality for bacterial infections due to its effectiveness and less likelihood of inducing bacterial resistance. Resistance to therapy is in part due to the ability of the organisms to form a biofilm, which provides a microenvironment that protects the microorganism from antibiotics and attack by the host's immune system. In vitro, PDI nonetheless was shown to be effective against Gram-negative and Gram-positive bacteria. When used in-vivo however, several factors were shown to influence and diminish the effectiveness of PDI, such as aggregation of PS and plasma protein binding. To circumvent these factors, different nanotechnology platforms were used to enhance the photodynamic inactivation efficacy, such as liposomes, micelles and nanoparticles, by reducing the PS aggregation and albumin binding to the PS. In general, studies have shown that photodynamic inactivation efficacy could be enhanced when suitable nanocarriers are used to deliver the PS.

Photodynamic effectiveness of laser diode combined with ozone to reduce STaphylicoccus aureus biofilm with exogenous chlorophyll of Dracaena angustifolia leaves

Photodynamic inactivation is an effective treatment that uses light irradiation, photosensitizer and oxygen. The aim of this study was to determine photodynamic effectiveness of laser diode combined with ozone to reduce Staphylococcus aureus biofilm using exogenous chlorophyll (Chlo). The chlorophyll was extracted from leave of Dracaena angustifolia. To determine the antibacterial effect of S. aureus biofilm treatments, samples were separated into Chlo, Laser, Chlo+Laser, Ozone, Ozone+Laser, Chlo+Ozone+Laser categories. The data were analyzed using ANOVA test. The result of this study showed that Chlo+Ozone+Laser combine treatment at 20 s exposure of ozone with 4 min of irradiation time lead to 80.26 % reduction of biofilm activity, which was the highest efficacy of all the treatment groups. The combination of laser, chlorophyll and lower ozone concentration increases the effectiveness of photodynamic inactivation.

Photodynamic Inactivation of Multidrug-Resistant Staphylococcus aureus Using Hybrid Photosensitizers Based on Amphiphilic Block Copolymer-Functionalized Gold Nanoparticles.

Multidrug-resistant Staphylococcus aureus (MRSA) has become one of the major causes of various infections, leading to morbidity in both healthy and immune-compromised populations worldwide. Herein, we report a novel type of hybrid photosensitizer based on amphiphilic block copolymer-functionalized gold nanoparticles. The design of the nanoparticles provides a facile means to incorporate hydrophobic photosensitizing molecules for use in aqueous media. The hybrid photosensitizers display greatly enhanced singlet oxygen generation and outstanding photodynamic inactivation (PDI) efficacy against MRSA under light illumination. These hybrid photosensitizers greatly improve the effectiveness of PDI against MRSA while not involving antibiotics.

Periopathogens differ in terms of the susceptibility to toluidine blue O-mediated photodynamic inactivation

BackgroundThe main goal of periodontal therapy is to eliminate the infection spreading in periodontium. Antimicrobial photodynamic therapy may be applied in order to eradicate pathogens remaining in periodontal tissues after conventional mechanical debridement, to improve the treatment results. The aim of this in vitro study was to evaluate the susceptibility of selected key periopathogens to toluidine blue O-mediated photodynamic inactivation and the influence of photosensitizer’s concentration and light dose on the effectiveness of this process. MethodsFollowing bacterial strains were used in the experiments: Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomyctemecomitans ATCC 33384, Fusobacterium nucleatum ATCC 10953. Toluidine blue O (TBO) was used in concentration ranging from 0.004 to 0.5mg/mL. Irradiation was performed by a non-laser red light source. ResultsComplete eradication of P. gingivalis was obtained upon the application of TBO in the concentration of 0.1mg/mL and the highest light dose. A, actinomycetemcomitans was, in turn, not susceptible to photodynamic inactivation regardless of the dosimetric parameters applied. High viability reductions were also obtained for F. nucleatum, however no complete eradication. The effectiveness of photodynamic inactivation of susceptible periopathogens was dependent on the light dose and photosensitizer’s concentration. ConclusionsPeriopathogens differ in terms of their susceptibility to photodynamic inactivation. Antimicrobial PDT may be valuable in the treatment of those cases of periodontal disease, in which P. gingivalis is a dominating pathogen. Microbiological examination prior to clinical application of aPDT may be recommended.

Photosensitizer in lipid nanoparticle: a nano-scaled approach to antibacterial function

Photosensitization-based antimicrobial therapy (PAT) is an alternative therapy aimed at achieving bacterial inactivation. Researchers use various photosensitizers to achieve bacterial inactivation. However, the most widely used approach involves the use of photosensitizers dispersed in aqueous solution, which could limit the effectiveness of photodynamic inactivation. Therefore, the approaches to encapsulate the photosensitizer in appropriate vehicles can enhance the delivery of the photosensitizer. Herein, Toluidine Blue O (TBO) was the photosensitizer, and lipid nanoparticles were used for its encapsulation. The lipid nanoparticle-based delivery system has been tailor-made for decreasing the average size and viscosity and increasing the formulation stability as well as the wettability of skin. Usage of an appropriate vehicle will also increase the cellular uptake of the photosensitizer into the bacterial cells, leading to the damage on cell membrane and genomic DNA. Evidence of effectiveness of the developed PAT on planktonic bacteria and biofilms was examined by fluorescence microscopy and scanning electron microscopy. Lipid nanoparticles protected the photosensitizer from aggregation and made the application easy on the skin as indicated in data of size distribution and contact angle. The use of lipid nanoparticles for encapsulating TBO could enhance photosensitization-based antimicrobial therapy as compared to the aqueous media for delivering photosensitizers.

Employment of methylene blue irradiated with laser light source in photodynamic inactivation of biofilm formed by Candida albicans strain resistant to fluconazole.

A promising approach for the eradication of biofilm formed by the yeast Candida albicans seems to be photodynamic inactivation (PDI). This work presents a use of methylene blue (MB, 1mM) irradiated with a red laser (output power 190mW/cm2, wavelength 660nm) for the eradication of a biofilm formed by the fluconazole-resistant (FLC-resistant) strain C. albicans CY 1123 compared to the standard strain C. albicans SC5314. The periods of irradiation corresponded to the fluence of 15, 23 and 57J/cm2. Effectiveness of PDI was evident with following percentage of survived biofilm cells: 24.57, 23.46, and 22.29% for SC5314 and 40.28, 17.91, and 5.89% for CY 1123, respectively, compared to the samples without irradiation. Light and confocal laser scanning microscopy confirmed the effectiveness of PDI. However, the morphological form of C. albicans seems to play an important role as well, since prolonged duration of irradiation did not increase efficiency of PDI on C. albicans SC5314. An experiment with the yeast-to-hyphae transition revealed that the FLC-resistant strain expressed a markedly reduced capacity to form hyphae compared to SC5314. We summarized that PDI was effective on biofilm formed by the FLC-resistant strain, but resistance most likely did not play significant role in PDI. Additionally, we observed differences in susceptibility to PDI between biofilms composed of the mycelia and only of the yeasts, and finally, the employment of a laser in PDI enabled a decreasing period of irradiation while maintaining the high effectiveness of PDI.

In vitro photoinactivation of bovine mastitis related pathogens

BackgroundBovine mastitis is considered the most important disease of worldwide dairy industry. Treatment of this disease is based on the application intramammary antibiotic, which favors an increase in the number of resistant bacteria in the last decade. Photodynamic inactivation (PDI) has been investigated in different areas of Health Sciences, and has shown great potential for inactivating different pathogens, without any selection of resistant microorganisms. The objective of this study was to investigate the efficacy of PDI in the inactivation of pathogens associated with bovine mastitis. MethodsWe tested the effectiveness of PDI against antibiotic resistant strains, isolated from bovine mastitis, from the following species: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, and the alga Prototheca zopfii. Nine experimental groups were evaluated: control, no treatment; light only, irradiation of a red light-emitting diode (λ=662 (20) nm) for 180s; exposure to 50μM methylene blue alone for 5min; and PDI for 5, 10, 30, 60, 120 and 180s. ResultsS. dysgalactiae, S. aureus, and C. bovis were inactivated after 30s of irradiation, whereas S. agalactiae was inactivated after 120s and P. zopfii at 180s of irradiation. ConclusionThese results show that PDI can be an interesting tool for inactivating pathogens for bovine mastitis.

In vitro photodynamic inactivation effects of Ru(II) complexes on clinical methicillin-resistant Staphylococcus aureus planktonic and biofilm cultures.

Photosensitizers (PSs) combined with light are able to generate antimicrobial effects. Ru(II) complexes have been recognized as a novel class of PSs. In this study, we investigated the effectiveness of photodynamic inactivation (PDI) mediated by three Ru(II) polypyridine complexes, 1-3, against four isolates of clinical methicillin-resistant Staphylococcus aureus (MRSA-1, MRSA-2, MRSA-3 and MRSA-4). In PDI of a planktonic culture of MRSA-1, compound 3 showed the highest efficacy, likely owing to its advantageous light absorption, (1) O2 quantum yield and bacterial cellular binding. The PDI efficacy of 3 was further evaluated against all other strains and MRSA-1 biofilms. At appropriate PS concentrations, viability reduction of 100% or 96.83% was observed in planktonic or biofilm forms of MRSA, respectively. The mechanisms of action were investigated using negative staining transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). It was demonstrated that PDI of planktonic bacteria was achieved primarily through damage to the cell envelope. Biofilms were eliminated through both the destruction of their structure and inactivation of the individual bacterial cells. In conclusion, Ru(II) complexes, especially 3, are potential candidates for the effective photodynamic control of MRSA infections.

Multiresistant Strains Are as Susceptible to Photodynamic Inactivation as Their Naïve Counterparts: Protoporphyrin IX-Mediated Photoinactivation Reveals Differences Between Methicillin-Resistant and Methicillin-SensitiveStaphylococcus aureusStrains

The current study was aimed at the investigation of differences in response to photoinactivation between methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolates. Moreover, we aimed to elucidate if the observed variation resulted from antimicrobial resistance mechanisms and strains' susceptibility to antibiotic therapy. Because of the emergence of multidrug resistance, the development of alternative antimicrobial strategies seems to be required. The concept of photodynamic inactivation (PDI) involves cell exposure to appropriate wavelength light that leads to the excitation of photosensitizer molecules, resulting in the production of reactive oxygen species responsible for cell inactivation and death. Recently, we have demonstrated a strain-dependent response of S. aureus to photoinactivation, and observed elevated resistance to PDI among MRSA strains. Nevertheless, the mechanism underlying this phenomenon remains unexplained. S. aureus response to protoporphyrin IX (PPIX)-mediated photoinactivation was studied for 424 MRSA/MSSA isolates. VITEK 2 Advanced Expert System was used to detect antimicrobial resistance mechanisms and strains' susceptibility to antibiotictherapy. Data obtained demonstrated that MRSA are significantly more resistant to photoinactivation than MSSA strains; however, the difference observed did not result from antimicrobial susceptibility or resistance mechanisms. Furthermore, regardless of the strains' origin, a similar effectiveness of PDI could be achieved. Moreover, it was determined that the ability to form biofilms in vitro, and the presence of mec element, does not explain the observed differences between MRSA and MSSA strains. PDI could be highly effective against multidrug resistant pathogens as well as their naïve counterparts. Nevertheless, regardless of the antimicrobial resistance mechanism, the difference in response to PDI between MRSA and MSSA exists.

Superoxide dismutase is upregulated in Staphylococcus aureus following protoporphyrin-mediated photodynamic inactivation and does not directly influence the response to photodynamic treatment.

BackgroundStaphylococcus aureus, a major human pathogen causes a wide range of disease syndromes. The most dangerous are methicillin-resistant S. aureus (MRSA) strains, resistant not only to all β-lactam antibiotics but also to other antimicrobials. An alarming increase in antibiotic resistance spreading among pathogenic bacteria inclines to search for alternative therapeutic options, for which resistance can not be developed easily. Among others, photodynamic inactivation (PDI) of S. aureus is a promising option. Photodynamic inactivation is based on a concept that a non toxic chemical, called a photosensitizer upon excitation with light of an appropriate wavelength is activated. As a consequence singlet oxygen and other reactive oxygen species (e.g. superoxide anion) are produced, which are responsible for the cytotoxic effect towards bacterial cells. As strain-dependence in photodynamic inactivation of S. aureus was observed, determination of the molecular marker(s) underlying the mechanism of the bacterial response to PDI treatment would be of great clinical importance. We examined the role of superoxide dismutases (Sod) in photodynamic inactivation of S. aureus as enzymes responsible for oxidative stress resistance.ResultsThe effectiveness of photodynamic inactivation towards S. aureus and its Sod isogenic mutants deprived of either of the two superoxide dismutase activities, namely SodA or SodM or both of them showed similar results, regardless of the Sod status in TSB medium. On the contrary, in the CL medium (without Mn++ ions) the double SodAM mutant was highly susceptible to photodynamic inactivation. Among 8 clinical isolates of S. aureus analyzed (4 MRSA and 4 MSSA), strains highly resistant and strains highly vulnerable to photodynamic inactivation were noticed. We observed that Sod activity as well as sodA and sodM transcript level increases after protoporphyrin IX-based photodynamic treatment but only in PDI-sensitive strains.ConclusionsWe confirmed that porphyrin-based photokilling efficacy is a strain-dependent phenomenon. We showed that oxidative stress sensitivity caused by the lack of both Sod enzymes can be relieved in the presence of Mn ions and partially in the presence of Fe ions. The fact that Sod activity increase is observed only in PDI-susceptible cells emphasizes that this is probably not a direct factor affecting S. aureus vulnerability to porphyrin-based PDI.

Photodynamic inactivation of free and vegetative bacteriophage T4

1. 1. Free bacteriophage T4 is inactivated when it is irradiated with visible light in the presence of methylene blue. Earlier evidence suggested that an internal component of the phage is an important target because penetration of the dye is important for the lethal effect. Photodynamic inactivation is pH-dependent owing to an effect of H + ions during irradiation rather than to their effect on penetration. Some irradiated phage can inject functional genetic material but lose their ability to form plaques, suggesting that their DNA is damaged. This was shown by marker rescue experiments with the rII gene; the gene was lost about two-thirds as rapidly as colony-forming ability. Loss of ability to inject is responsible for much of this ‘long-range hit’ effectiveness of photodynamic inactivation. The results suggest that 2 similarly sensitive targets exist in the free phage: the injection apparatus and DNA. 2. 2. Plaque formation by infected bacteria is initially nearly as sensitive as inactivation of free phages. DNA and protein synthesis are severely impaired. Contrary to the well known development of resistance to ultraviolet irradiation, X-rays of 32P decay, sensitivity to photodynamic inactivation does not change during early development. When rII mutants are used, resistance to photodynamic inactivation appears about 9 min after infection, or just before completed intracellular phages are formed. However, sensitivity of bacteria infected with r + phage persists till about 15 min. In this case, the release of intact phage might be blocked, or the intracellular phage might be in a sensitive state. These results demonstrate another difference between rII and r +, perhaps one which permits lysis inhibition by photodynamic inactivation after infection with r +.