Influence of sucrose on growth and sensitivity of <Emphasis Type="Italic">Candida albicans</Emphasis> alone and in combination with <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> and <Emphasis Type="Italic">Streptococcus mutans</Emphasis> to photodynamic therapy

This study has evaluated the effects of photodynamic inactivation (PDI) using erythrosine as photosensitizer and green light-emitting diode (LED) on biofilms of Candida albicans alone and in combination with Enterococcus faecalis and Streptococcus mutans. We have also evaluated the effect of sucrose on biofilm formation and bacterial growth and sensitivity to PDI. Biofilms were formed in suspension of 10⁶ cells/ml on plates before being grown in broth culture with and without sucrose and incubated for 48 h. Next, the treatment was applied using erythrosine at a concentration of 400 μM for 5 min and green LED (532 ± 10 nm) for 3 min on biofilms alone and in combination. The plates were washed and sonicated to disperse the biofilms, and serial dilutions were carried and aliquots seeded in Sabouraud agar before incubation for 48 h. Next, the colony-forming units per milliliter (CFU/ml; log₁₀) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Results show that S. mutans favors the growth of C. albicans in biofilms with sucrose, with treatment not being effective. However, when the biofilm was grown without sucrose, we found a reduction in biofilm formation and a significant decrease in the PDI treatment (P < 0.0001). In conclusion, both growth and sensitivity to PDI in biofilms of C. albicans are strongly influenced by bacterial combination, and the presence of sucrose affected directly the growth and sensitivity of the biofilm to PDI as sucrose is the substrate for construction of the exopolysaccharide matrix.     

Influence of pre-irradiation time employed in antimicrobial photodynamic therapy with diode laser

The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were as follows: times of pre-irradiation of the photosensitizer in three levels (1, 2, or 5 min). For the control of the cariogenic dental biofilm with antimicrobial photodynamic therapy (aPDT), methylene blue (0.01%) was used in association with the diode laser (InGaAlP) with a wavelength of 660 nm. Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into five groups and 15 C. albicans cultures, also divided into five groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony-forming units (CFU) per square millimeter of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey’s post-test. All analyses were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p < 0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans, there was no statistical difference between the groups (p > 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 min can be used to reduce the microbial load of S. mutans.     

Enhancement of photodynamic inactivation of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms by disruptive strategies

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm²) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.     

Antimicrobial photodynamic therapy—a promising treatment for prosthetic joint infections

Periprosthetic joint infection (PJI) is associated with high patient morbidity and a large financial cost. This study investigated Photodynamic Therapy (PDT) as a means of eradicating bacteria that cause PJI, using a laser with a 665-nm wavelength and methylene blue (MB) as the photosensitizer. The effectiveness of MB concentration on the growth inhibition of methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Pseudomonas aeruginosa and Acinetobacter baumannii was investigated. The effect of laser dose was also investigated and the optimized PDT method was used to investigate its bactericidal effect on species within planktonic culture and following the formation of a biofilm on polished titanium and hydroxyapatite coated titanium discs. Results showed that Staphylococci were eradicated at the lowest concentration of 0.1 mM methylene blue (MB). With P. aeruginosa and A. baumannii, increasing the MB concentration improved the bactericidal effect. When the laser dose was increased, results showed that the higher the power of the laser the more bacteria were eradicated with a laser power?≥?35 J/cm² and an irradiance of 35 mW/cm², eradicating all S. epidermidis. The optimized PDT method had a significant bactericidal effect against planktonic MRSA and S. epidermidis compared to MB alone, laser alone, or control (no treatment). When biofilms were formed, PDT treatment had a significantly higher bactericidal effect than MB alone and laser alone for all species of bacteria investigated on the polished disc surfaces. P. aeruginosa grown in a biofilm was shown to be less sensitive to PDT when compared to Staphylococci, and a HA-coated surface reduced the effectiveness of PDT. This study demonstrated that PDT is effective for killing bacteria that cause PJI.     

Curcumin-mediated anti-microbial photodynamic therapy against <Emphasis Type="Italic">Candida dubliniensis</Emphasis> biofilms

The purpose of this study was to evaluate the effectiveness of anti-microbial photodynamic therapy (aPDT) mediated by curcumin (Cur) associated with LED light against biofilms of Candida dubliniensis, and further, investigate cellular uptake and drug penetration through the biofilms under confocal laser scanning microscopy (CLSM). Four C. dubliniensis strains were tested: three clinical isolates from HIV-positive patients and one reference strain (CBS 7987). Biofilms were treated with three Cur concentrations (20.0, 30.0, and 40.0 μM). All samples were incubated in the dark for 20 min and exposed to a 5.28 J/cm² of LED light fluence. Additional samples of each strain were treated either with Cur or LED light only. Control samples had neither Cur nor light. After aPDT, results were read using the XTT salt reduction method. The data were statistically analyzed by two-way ANOVA followed by Games-Howell post-hoc test (α?=?0.05). Confocal laser scanning microscopy was used to verify both the uptake of Cur by yeast cells and its penetration through the biofilm. The results showed that aPDT promoted significant reduction on the metabolism of the biofilm-organized cells of C. dubliniensis. Further, while Cur was rapidly taken up by C. dubliniensis cells, a longer time interval was required to allow Cur penetration into biofilm cells. Based on these results, aPDT associating LED and Cur presents promising potential on fungal control of biofilms of C. dubliniensis.     

Photoinactivation effect of eosin methylene blue and chlorophyllin sodium-copper against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of eosin methylene blue according to Giemsa as photosensitizer is presented for the first time in this paper. The present study evaluated the potential application of chlorophyllin sodium copper salt (CuChlNa) and eosin methylene blue according to Giemsa (EMB) as antimicrobial photosensitizers (aPS) for photodynamic inactivation (PDI) of Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria. The experiments were performed using S. aureus stain ATCC 25923 and E. coli ATCC 25922 in which five aPS concentrations (0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μM for S. aureus and 0.0, 5.0, 10.0, 20.0, 40.0, and 50.0 μM for E. coli) were prepared and added in 2 mL of a saline solution containing the bacterial inoculum. After aPS incubation, the samples were divided into two groups, one kept in the dark and another submitted to the illumination. Then, the bacterial inactivation was determined 18 h after the incubation at 37 °C by counting the colony-forming units (CFU). The results revealed that both EMB and CuChlNa can be used as aPS for the photoinactivation of S. aureus, while only EMB was able to photoinactivate E. coli. Nevertheless, a more complex experimental setup was needed for photoinactivation of E. coli. The data showed that EMB and CuChlNa presented similar photoinactivation effects on S. aureus, in which bacterial growth was completely inhibited at photosensitizer (PS) concentrations over 5 μM, when samples were previously incubated for 30 min and irradiated by a light dose of 30 J cm^?2 as a result of an illumination of 1 h at 8.3 mW cm^?2 by using a red light at 625 nm with a 1 cm beam diameter and output power of 6.5 mW. In the case of E. coli, bacterial growth was completely inhibited only when combining a PS incubation period of 120 min with concentrations over 20 μM.     

The impact of antimicrobial photodynamic therapy in an artificial biofilm model

The susceptibility of bacterial cultures in biofilm formations is important for a variety of clinical treatment procedures. Therefore, the aim of the study was to assess the impact of laser-induced antimicrobial photodynamic therapy on the viability of Streptococcus mutans cells employing an artificial biofilm model. Using sterile chambered coverglasses, a salivary pellicle layer was formed in 40 chambers. Streptococcus mutans cells were inoculated in a sterile culture medium. Employing a live/dead bacterial viability kit, bacteria with intact cell membranes stained fluorescent green. Each pellicle-coated test chamber was filled with 0.7 ml of the bacterial suspension and analysed using a confocal laser scanning microscope within a layer of 10 μm at intervals of 1 μm from the pellicle layer. Phenothiazine chloride was used as a photosensitizer in all 40 test chambers. A diode laser (wavelength 660 nm, output power 100 mW) was used to irradiated 20 chambers for 2 min. Fluorescence values in the test chambers after laser irradiation (median 2.1 U, range 0.4–3.4 U) were significantly lower than baseline values after adding the photosensitizer (median 3.6 U, range 1.1–9.0; p?p?>?0.05). The present study indicated that laser irradiation is an essential part of antimicrobial photodynamic therapy to reduce bacteria within a layer of 10 μm. Further studies are needed to evaluate the maximum biofilm thickness that still allows a toxic effect on microorganisms.     

Effects of photodynamic laser and violet-blue led irradiation on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm and <Emphasis Type="Italic">Escherichia coli</Emphasis> lipopolysaccharide attached to moderately rough titanium surface: in vitro study

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This study investigates the in vitro efficacy of photodynamic treatment (PDT) with methylene blue (MB) photoactivated with λ 635 nm diode laser and of λ 405 nm violet-blue LED phototreatment for the reduction of bacterial biofilm and lipopolysaccharide (LPS) adherent to titanium surface mimicking the bone-implant interface. Staphylococcus aureus biofilm grown on titanium discs with a moderately rough surface was subjected to either PDT (0.1% MB and λ 635 nm diode laser) or λ 405 nm LED phototreatment for 1 and 5 min. Bactericidal effect was evaluated by vital staining and residual colony-forming unit count. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, discs coated with Escherichia coli LPS were treated as above before seeding with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Both PDT and LED phototreatment induced a statistically significant (p?<?0.05 or higher) reduction of viable bacteria, up to ?99 and ?98% (5 min), respectively. Moreover, besides bactericidal effect, PDT and LED phototreatment also inhibited LPS bioactivity, assayed as nitrite formation, up to ?42%, thereby blunting host inflammatory response. Non-invasive phototherapy emerges as an attractive alternative in the treatment of peri-implantitis to reduce bacteria and LPS adherent to titanium implant surface without causing damage of surface microstructure. Its efficacy in the clinical setting remains to be investigated.     

The effects of diode laser on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm and <Emphasis Type="Italic">Escherichia coli</Emphasis> lipopolysaccharide adherent to titanium oxide surface of dental implants. An in vitro study

Effective decontamination of biofilm and bacterial toxins from the surface of dental implants is a yet unresolved issue. This in vitro study aims at providing the experimental basis for possible use of diode laser (λ 808 nm) in the treatment of peri-implantitis. Staphylococcus aureus biofilm was grown for 48 h on titanium discs with porous surface corresponding to the bone-implant interface and then irradiated with a diode laser (λ 808 nm) in noncontact mode with airflow cooling for 1 min using a Ø 600-μm fiber. Setting parameters were 2 W (400 J/cm²) for continuous wave mode; 22 μJ, 20 kHz, 7 μs (88 J/cm²) for pulsed wave mode. Bactericidal effect was evaluated using fluorescence microscopy and counting the residual colony-forming units. Biofilm and titanium surface morphology were analyzed by scanning electron microscopy (SEM). In parallel experiments, the titanium discs were coated with Escherichia coli lipopolysaccharide (LPS), laser-irradiated and seeded with RAW 264.7 macrophages to quantify LPS-driven inflammatory cell activation by measuring the enhanced generation of nitric oxide (NO). Diode laser irradiation in both continuous and pulsed modes induced a statistically significant reduction of viable bacteria and nitrite levels. These results indicate that in addition to its bactericidal effect laser irradiation can also inhibit LPS-induced macrophage activation and thus blunt the inflammatory response. The λ 808-nm diode laser emerges as a valuable tool for decontamination/detoxification of the titanium implant surface and may be used in the treatment of peri-implantitis.     

Red and infrared laser therapy inhibits in vitro growth of major bacterial species that commonly colonize skin ulcers

Low-level laser therapy (LLLT) is used in chronic wounds due to its healing effects. However, bacterial species may colonize these wounds and the optimal parameters for effective bacterial inhibition are not clear. The aim of this study was to analyze the effect of LLLT on bacterial growth in vitro. Bacterial strains including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were suspended in saline solution at a concentration of 10³ cells/ml and exposed to laser irradiation at wavelengths of 660, 830, and 904 nm at fluences of 0 (control), 3, 6, 12, 18, and 24 J/cm². An aliquot of the irradiated suspension was spread on the surface of petri plates and incubated at 37 °C for quantification of colony-forming unit after 24, 48, and 72 h. Laser irradiation inhibited the growth of S. aureus at all wavelengths and fluences higher than 12 J/cm², showing a strong correlation between increase in fluence and bacterial inhibition. However, for P. aeruginosa, LLLT inhibited growth at all wavelengths only at a fluence of 24 J/cm². E. coli had similar growth inhibition at a wavelength of 830 nm at fluences of 3, 6, 12, and 24 J/cm². At wavelengths of 660 and 904 nm, growth inhibition was only observed at fluences of 12 and 18 J/cm², respectively. LLLT inhibited bacterial growth at all wavelengths, for a maximum of 72 h after irradiation, indicating a correlation between bacterial species, fluence, and wavelength.     

Repeated applications of photodynamic therapy on <Emphasis Type="Italic">Candida glabrata</Emphasis> biofilms formed in acrylic resin polymerized

Previous studies have been suggested that photodynamic therapy (PDT) can be used as an adjuvant treatment for denture stomatitis. In this study, we evaluated the effects of multiple sessions of PDT on Candida glabrata biofilms in specimens of polymerized acrylic resin formed after 5 days. Subsequently, four applications of PDT were performed on biofilms in 24-h intervals (days 6–9). Also, we evaluated two types of PDT, including application of laser and methylene blue or light-emitting diode (LED) and erythrosine. The control groups were treated with physiological solution. The effects of PDT on biofilm were evaluated after the first and fourth application of PDT. The biofilm analysis was performed by counting the colony-forming units. The results showed that between the days 6 and 9, the biofilms not treated by PDT had an increase of 5.53 to 6.05 log (p?=?0.0271). Regarding the treatments, after one application of PDT, the biofilms decreased from 5.53 to 0.89 log. When it was done four applications, the microbial reduction ranged from 6.05 log to 0.11 log. We observed that one application of PDT with laser or LED caused a reduction of 3.36 and 4.64 compared to the control groups, respectively (p?=?0.1708). When it was done four applications of PDT, the reductions achieved were 1.57 for laser and 5.94 for LED (p?=?0.0001). It was concluded that repeated applications of PDT on C. glabrata biofilms showed higher antimicrobial activity compared to single application. PDT mediated by LED and erythrosine was more efficient than the PDT mediated by laser and methylene blue.     

Photodynamic antimicrobial therapy of curcumin in biofilms and carious dentine

Photodynamic therapy (PDT) is a technique that involves the activation of photosensitizers by light in the presence of oxygen, resulting in the production of reactive radicals that are capable of inducing cell death. The present study evaluated the susceptibility of Streptococcus mutans and Lactobacillus acidophilus to PDT grown as multi-species in the biofilm phase versus in dentine carious lesions. A brain-heart infusion culture medium supplemented with 1 % glucose, 2 % sucrose, and 1 % young primary culture of L. acidophilus 10⁸ CFU/mL and S. mutans 10⁸ CFU/mL was used to develop multi-species biofilms and to induce caries on human dentine slabs. Five different concentrations of curcumin (0.75, 1.5, 3.0, 4.0, and 5.0 g/L) were used associated with 5.7 J/cm² light emission diode. Four different groups were analyzed L?D? (control group), L?D+ (drug group), L+D? (light group), and L+D+ (PDT group). ANOVA/Tukey’s tests were conducted to compare groups. A significant reduction (p <0.05) in cell viability was observed in the biofilm phase following photosensitization with all curcumin concentrations tested. To achieve significant bacterial reduction (p <0.05) in carious dentine, it was necessary to utilize 5.0 g/L of curcumin in association with blue light. No significant reduction was found for L?D+, supporting the absence of the drug’s dark toxicity. S. mutans and L. acidophilus were susceptible to curcumin in the presence of blue light. However, due to light penetration and drug diffusion difficulties, these microorganisms within dentine carious lesions were less affected than they were in the biofilm phase.     

Photodynamic antimicrobial chemotherapy (PACT) using toluidine blue inhibits both growth and biofilm formation by <Emphasis Type="Italic">Candida krusei</Emphasis>

Among non-albicans Candida species, the opportunistic pathogen Candida krusei emerges because of the high mortality related to infections produced by this yeast. The Candida krusei is an opportunistic pathogen presenting an intrinsic resistance to fluconazol. In spite of the reduced number of infections produced by C. krusei, its occurrence is increasing in some groups of patients submitted to the use of fluconazol for prophylaxis. Photodynamic antimicrobial chemotherapy (PACT) is a potential antimicrobial therapy that combines visible light and a nontoxic dye, known as a photosensitizer, producing reactive oxygen species (ROS) that can kill the treated cells. The objective of this study was to investigate the effects of PACT, using toluidine blue, as a photosensitizer on both growth and biofilm formation by Candida krusei. In this work, we studied the effect of the PACT, using TB on both cell growth and biofilm formation by C. krusei. PACT was performed using a light source with output power of 0.068 W and peak wavelength of 630 nm, resulting in a fluence of 20, 30, or 40 J/cm². In addition, ROS production was determined after PACT. The number of samples used in this study varied from 6 to 8. Statistical differences were evaluated by analysis of variance (ANOVA) and post hoc comparison with Tukey-Kramer test. PACT inhibited both growth and biofilm formation by C. krusei. It was also observed that PACT stimulated ROS production. Comparing to cells not irradiated, irradiation was able to increase ROS production in 11.43, 6.27, and 4.37 times, in the presence of TB 0.01, 0.02, and 0.05 mg/mL, respectively. These results suggest that the inhibition observed in the cell growth after PACT could be related to the ROS production, promoting cellular damage. Taken together, these results demonstrated the ability of PACT reducing both cell growth and biofilm formation by C. krusei.     

Virulence factors of fluconazole-susceptible and fluconazole-resistant <Emphasis Type="Italic">Candida albicans</Emphasis> after antimicrobial photodynamic therapy

This study evaluated the effects of antimicrobial photodynamic therapy (aPDT) mediated by Photodithazine® (PDZ) and LED light on the virulence factors of fluconazole-susceptible (CaS) and fluconazole-resistant (CaR) Candida albicans. Standardized suspensions of strains were prepared (10⁷), and after 48 h of biofilm formation, these strains were incubated with PDZ (100 mg/L) for 20 min and exposed to LED light (660 nm, 37.5 J/cm²). Additional samples were treated with PDZ or light only, and the control consisted of biofilms that received no treatment. After aPDT, the cells were recovered and the virulence factors were evaluated. To analyze the capacity of adhesion, cells were recovered after aPDT and submitted to the adhesion process in the bottom of a 96-well plate. After this, metabolic activity tests (XTT assay) and cell viability (colony forming units per milliliter, CFU/mL) were applied. To evaluate the biofilm-forming ability after aPDT, the cells recovered were submitted to biofilm formation procedures, and the biofilm formed was evaluated by XTT, CFU/mL, and total biomass (crystal violet) tests. Lastly, the capacity for synthesizing protease and phospholipase enzymes after aPDT was evaluated by fluorimetric tests. Data were analyzed by two- or three-way ANOVA tests (p?≤?0.05). It was verified that aPDT reduced the viability of both strains, fluconazole-susceptible and fluconazole-resistant C. albicans. It was also observed that the CaR strain had lower susceptibility to the aPDT when compared with the CaS strain. However, regarding the virulence factors evaluated, it was demonstrated that aPDT did not alter the adherence and biofilm formation ability and enzymatic production.     

Action of antimicrobial photodynamic therapy on heterotypic biofilm: <Emphasis Type="Italic">Candida albicans</Emphasis> and <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>

The increase in survival and resistance of microorganisms organized in biofilms demonstrates the need for new studies to develop therapies able to break this barrier, such as photodynamic therapy, which is characterized as an alternative, effective, and non-invasive treatment. The objective was to evaluate in vitro the effect of antimicrobial photodynamic therapy on heterotypic biofilms of Candida albicans and Bacillus atrophaeus using rose bengal (12.5 μM) and light-emitting diode (LED) (532 nm and 16.2 J). We used standard strains of B. atrophaeus (ATCC 9372) and C. albicans (ATCC 18804). The biofilm was formed in the bottom of the plate for 48 h. For the photodynamic therapy (PDT) experimental groups, we added 100 μL of rose bengal with LED (P+L+), 100 μL of rose bengal without LED (P+L?), 100 μL of NaCl 0.9 % solution with LED (P?L+), and a control group without photosensitizer or LED (P?L?). The plates remained in agitation for 5 min (pre-irradiation) and were irradiated with LED for 3 min, and the biofilm was detached using an ultrasonic homogenizer for 30 s. Serial dilutions were plated in BHI agar and HiChrom agar and incubated at 37 °C/48 h. There was a reduction of 33.92 and 29.31 % of colony-forming units per milliliter (CFU/mL) for C. albicans and B. atrophaeus, respectively, from the control group to the group subjected to PDT. However, statistically significant differences were not observed among the P+L+, P+L?, P?L+, and P?L? groups. These results suggest that antimicrobial photodynamic therapy using rose bengal (12.5 μM) with a pre-irradiation period of 5 min and LED for 3 min was not enough to cause a significant reduction in the heterotypic biofilms of C. albicans and B. atrophaeus.     

Photodynamic inactivation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> biofilms and planktonic cells by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester

The treatment of Klebsiella pneumoniae, particularly extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae, is currently a great challenge. Photodynamic antimicrobial chemotherapy is a promising approach for killing antibiotic-resistant bacteria. The aim of this study was to evaluate the capacity of 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL) in the presence of white light to cause photodynamic inactivation (PDI) of K. pneumoniae planktonic and biofilm cells. In the presence of white light, 5-ALA and MAL inactivated planktonic cells in a concentration-dependent manner. Biofilms were also sensitive to 5-ALA and MAL-mediated PDI. The mechanisms by which 5-ALA and MAL caused PDI of ESBL-producing K. pneumonia were also investigated. Exposure of K. pneumonia to light in the presence of either 5-ALA or MAL induced cleavage of genomic DNA and the rapid release of intracellular biopolymers. Intensely denatured cytoplasmic contents and aggregated ribosomes were also detected by transmission electron microscopy. Scanning electron microscopy showed that PDI of biofilms caused aggregated bacteria to detach and that the bacterial cell envelope was damaged. This study provides insights into 5-ALA and MAL-mediated PDI of ESBL-producing K. pneumoniae.     

Enhancement of photo-bactericidal effect of tetrasulfonated hydroxyaluminum phthalocyanine on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

At the present time, photodynamic inactivation (PDI) is receiving considerable interest for its potential as an antimicrobial therapy. The results of our study indicate that enhancement of the phototoxic effect on Pseudomonas aeruginosa can be achieved by combination of tetrasulfonated hydroxyaluminum phthalocyanine (AlPcS₄) and bimetallic gold/silver nanoparticles (Au/Ag-NPs) synthesized by the cell-free filtrate of Aureobasidium pullulans. The bimetallic nanoparticles were characterized by a number of techniques including UV-vis, XPS, TEM, and SEM-EDS to be 14 ± 3 nm spherical particles coated with proteins. The effect of diode lasers with the peak-power wavelength ? = 650 nm (output power of 10 and 40 mW; radiation intensity of 26 and 105 mW/cm²) in combination with the AlPcS₄ and the bimetallic nanoparticles mixture on the viability of P. aeruginosa rods was shown. Particularly high efficiency of killing bacterial cells was obtained for the light intensity of 105 mW/cm², after 20, 30, and 40 min of irradiation corresponding to 126, 189^, and 252 J/cm² energy fluences. For AlPcS₄+Au/Ag-NPs treatment, the viable count reduction were equal to 99.90, 99.96, and 99.975%, respectively. These results were significantly better than those accomplished for irradiated separated assays of AlPcS₄ and Au/Ag-NPs.     

Photodynamic inactivation of pathogenic species <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Candida albicans</Emphasis> with lutetium (III) acetate phthalocyanines and specific light irradiation

Photodynamic inactivation (PDI) is a light-associated therapeutic approach suitable for treatment of local acute infections. The method is based on specific light-activated compound which by specific irradiation and in the presence of molecular oxygen produced molecular singlet oxygen and other reactive oxygen species, all toxic for pathogenic microbial cells. The study presents photodynamic impact of two recently synthesized water-soluble cationic lutetium (III) acetate phthalocyanines (LuPc-5 and LuPc-6) towards two pathogenic strains, namely, the Gram-negative bacterium Pseudomonas aeruginosa and a fungus Candida albicans. The photodynamic effect was evaluated for the cells in suspensions and organized in 48-h developed biofilms. The relatively high levels of uptakes of LuPc-5 and LuPc-6 were determined for fungal cells compared to bacterial cells. The penetration depths and distribution of both LuPcs into microbial biofilms were investigated by means of confocal fluorescence microscopy. The photoinactivation efficiency was studied for a wide concentration range (0.85–30 μM) of LuPc-5 and LuPc-6 at a light dose of 50 J cm^?2 from red light-emitting diode (LED; 665 nm). The PDI study on microbial biofilms showed incomplete photoinactivation (<3 logs) for the used gentle drug-light protocol.     

Time-dependent antimicrobial effect of photodynamic therapy with TONS 504 on <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa (P. aeruginosa) is a major cause of infectious keratitis, which itself is a major cause of blindness worldwide. We have now evaluated the time-dependent effectiveness of photodynamic antimicrobial chemotherapy (PACT) with the chlorin derivative TONS 504 and a light-emitting diode (LED) on P. aeruginosa in vitro. PACT with TONS 504 (10 mg/L) and irradiation (30 J/m²) by an LED device that delivers light centered on a wavelength of 660 nm was applied to 1?×?10⁶ colony-forming units of P. aeruginosa in liquid medium. The bacteria were then cultured at 37 °C for various times before assay of viability by determination of colony formation on agar plates. The effect of a second irradiation at 3 h after the initial LED exposure was also examined. Bacterial growth was markedly inhibited between 3 and 9 h after PACT with TONS 504, with the maximal effect being apparent at 3 h. Furthermore, a second exposure to LED irradiation at 3 h after the first treatment enhanced the inhibitory effect on bacterial growth. PACT with TONS 504 thus inhibited the growth of P. aeruginosa in a time-dependent manner, and an additional irradiation exposure applied 3 h after the first LED treatment greatly increased the effectiveness of PACT. This antibacterial system thus warrants further evaluation with regard to its potential effectiveness for the treatment of infectious keratitis.     

Preclinical study of a cost-effective photodynamic therapy protocol for treating oral candidoses

Photodynamic therapy (PDT) is a promising treatment for oral candidoses. Its use as an alternative to antifungals prevents several adverse effects, including microbial resistance. However, most PDT protocols do not employ devices and consumables commonly available in dental practice, thus influencing treatment affordability. This study aimed to determine the efficacy of a PDT method based on light curing units’ blue LEDs combined to a plaque-disclosing composition (5% erythrosine) against C. albicans in culture and in a murine model of oral candidosis. Standard and resistant fungal strains were tested in vitro in planktonic and biofilm forms. PDT (pre-irradiation time periods: 30 and 60 s; irradiation time: 3 min) was compared to control conditions without light and/or erythrosine. Mice with induced oral candidosis (n = 40) randomly received PDT or similar control conditions with subsequent C. albicans count. These mice underwent histological analysis, as well as 12 healthy mice submitted to experimental treatments. PDT completely inactivated C. albicans planktonic cells and biofilm. Control conditions presented minor differences (ANOVA, p < 0.05), with mean values ranging from 5.2 to 6.8 log10 (UFC/mL). Infected mice presented no significant difference in C. albicans counts consequent to treatments (ANOVA, p = 0.721), although the PDT protocol was able to enhance the inflammatory infiltrate in healthy mice. It can be concluded that the tested PDT protocol can inactivate C. albicans but still needs further investigation in order to achieve efficacy and safety.