The oxidative stress and metabolic response of Acinetobacter baumannii for aPDT multiple photosensitization.

The use of antimicrobial photodynamic inactivation as a non-antibiotic alternative method to inactivate Acinetobacter baumannii was described in response to the ever-growing problem of antibiotic resistance. It was found that irradiation of the bacterial suspension for 10 min reduced the number of viable cells by approximately 99% and this energy fluence was considered to be sub-lethal phototherapy. The lethal dose of laser light (cell mortality about 99.9%) was 9.54 J cm-2, which corresponds to 30 min of irradiation. After a 15-fold phototherapy cycle, the tolerance to aPDT decreased, resulting in a decrease in the number of viable cells by 2.15 and 3.23 log10 CFU/ml units with the use of sub-lethal and lethal light doses, respectively. Multiple photosensitizations decreased the biofilm formation efficiency by 25 ± 1% and 35 ± 1%, respectively. No changes in antibiotic resistance were observed, whereas the cells were more sensitive to hydrogen peroxide. Metabolomic changes after multiple photosensitization were studied and 1H NMR measurements were used in statistical and multivariate data analysis. Many significant changes in the levels of the metabolites were detected demonstrating the response of A. baumannii to oxidative stress.

Biogenic Gold Nanoparticles Decrease Methylene Blue Photobleaching and Enhance Antimicrobial Photodynamic Therapy.

Antibiotic resistance is a growing concern that is driving the exploration of alternative ways of killing bacteria. Here we show that gold nanoparticles synthesized by the mycelium of Mucor plumbeus are an effective medium for antimicrobial photodynamic therapy (PDT). These particles are spherical in shape, uniformly distributed without any significant agglomeration, and show a single plasmon band at 522-523 nm. The nanoparticle sizes range from 13 to 25 nm, and possess an average size of 17 ± 4 nm. In PDT, light (from a source consisting of nine LEDs with a peak wavelength of 640 nm and FWMH 20 nm arranged in a 3 × 3 array), a photosensitiser (methylene blue), and oxygen are used to kill undesired cells. We show that the biogenic nanoparticles enhance the effectiveness of the photosensitiser, methylene blue, and so can be used to kill both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The enhanced effectiveness means that we could kill these bacteria with a simple, small LED-based light source. We show that the biogenic gold nanoparticles prevent fast photobleaching, thereby enhancing the photoactivity of the methylene blue (MB) molecules and their bactericidal effect.

Sulfonated hydroxyaluminum phthalocyanine-biogenic Au/Ag alloy nanoparticles mixtures for effective photo-eradication of Candida albicans.

In response to the increasingly widespread resistance of fungi to traditional treatment, we have reported successful photodynamic inactivation of Candida albicans planktonic cells using di-(AlPcS2) and trisulfonated (AlPcS3) hydroxyaluminum phthalocyanines in combination with Au/Ag alloy nanoparticles synthesized by the cell-free filtrate of Trichoderma koningii. These nanostructures with Au:Ag molar ratios 2:1, 1:1 and 1:2 have individual plasmonic band at 513-515 nm, 505-509 nm and 486-489 nm, respectively. XPS analysis of the ratio of gold to silver on the surface of these alloys indicated that Au and Ag formed a bimetallic system, wherein Au was coated with Ag. The XRD pattern revealed the angles at 38.2, 44.5, 64.9 and 78.0°. TEM analysis indicated that the average diameter of the synthesized alloys was 9 ± 3 nm, 8 ± 3 nm and 16 ± 3 nm for structures with Au:Ag molar ratios 1:1, 1:2 and 2:1, respectively. The FTIR band absorption, SEM-EDS analysis and basic elemental composition obtained by XPS confirmed that these nanostructures are stabilized by protein(s). Diode laser with the peak-power wavelength ʎ = 650 nm (output power of 40 mW; power density of 105 mW cm-2) was used as a light source. The mixture of AlPcS2+Au/Ag-NPs (Au:Ag = 2:1) can be considered as an effective photosensitizer, because eradication of C. albicans, as required by the American Society of Microbiology (99.9 %), was achieved at a low dose of light of 31.5 J cm-2. It was postulated that this low dose of light applied to the photo-induced fungicidal effect may be painless for potential patients.

The effect of glucose and human serum on 5-aminolevulinic acid mediated photodynamic inactivation of Candida albicans.

In response to the growing number of life-threatening infections caused by opportunistic Candida albicans fungi we have examined and discussed the effect of glucose and human serum on the efficiency of photosensitization of C. albicans cells with 5-aminolevulinic acid (5-ALA) used as a precursor of protoporphyrin IX (PpIX). We used 630 nm laser diode as an excitation source to enhance the penetration depth and diminish unwanted light scattering, additionally the experiments were performed in planktonic and biofilm cultures to differentiate the results obtained for cells showing various phenotypic characteristics. Based on performed experiments it became obvious that the incubation of C. albicans fungal cells with 5-ALA in the presence of glucose caused a significant increase in concentration of intracellular PpIX, resulting in a higher efficacy of photo-toxic effect. The observed results were rationalized by the role of glucose as an energy source, which supported the active transport of 5-ALA into cells. In contrary to glucose, the presence of human serum caused a significant reduction in PpIX concentration in C. albicans cells with exogenous delivery of 5-ALA, resulting in a less efficient photo-fungicidal effect. In general, the obtained results contributed to the current search for efficient, light activated anti-fungal treatments of high efficiency.

Synergistic effect of methylene blue and biogenic gold nanoparticles against Enterococcus faecalis.

This study reports successful photodynamic inactivation of planktonic and biofilm cells of Enterococcus faecalis using Methylene Blue (MB) in combination with gold nanoparticles synthesized using the cell-free filtrate obtained from 3-day biomass of Trichoderma asperellum strain. Monodispersed colloidal gold nanoparticles were characterized by UV-vis absorption, TEM and DLS to be 13 ±â€¯3 nm spheres. Diode lasers with the peak-power wavelength ʎ = 660 nm (output power of 21, 41 and 68 mW; power density of 55, 108 and 179 mW∙cm-2, respectively, were used as a light source to study the effects of MB alone, the gold nanoparticles alone (AuNPs) and the MB + AuNPs mixture on the viability of E. faecalis cells. The lethal effect of planktonic cells was achieved for MB after 30 min of laser irradiation with energy fluence of 322 J∙cm-2. When MB + AuNPs mixture was used as photosensitizer, the lethal effect was achieved with energy fluence of 292 J∙cm-2. The biofilm culture was more resistant to photo-inactivation and the best bactericidal effect of MB as photosensitizer was found after light dose of 483 J∙cm-2. The bacterial cell viability was reduced by 99.92%. It was proved that MB + AuNPs mixture synergistically enhances the kill of the studied microorganism as the same light dose resulted in 99.991% kill.

The possible mechanism of the formation of silver nanoparticles by Penicillium cyclopium.

This contribution describes the biomineralization of silver nanoparticles by the microbial reduction of Ag (I) ions using the mycelium and the cell-free extract of Penicillium cyclopium. Different techniques, such as UV-Vis, SEM, TEM, FT-IR and GPC were used to characterize the obtained nanoparticles and understand the mechanism of their biosynthesis. The SEM and TEM images demonstrated the presence of silver nanoparticles on the mycelia surface suggesting that these particles are synthesized on the fungal cell wall. FT-IR analysis of the mycelium revealed two main types of compounds (saccharides and proteins) and these molecules might be involved in the formation of silver nanoparticles on the surface of mycelium. Ultraviolet-visible spectroscopy and TEM analysis confirmed the formation of silver nanoparticles with different shapes by the cell-free extract of P. cyclopium. Their size ranges from 12 to 25 nm and possess an average size of 16 ±â€¯6 nm. GPC analysis of this filtrate revealed a few peaks responsible for polysaccharides and proteins presence. The only protein fraction with the mass approximately to 5000 Da indicated the formation of silver nanoparticles. Polypeptide(s) as the major molecules involved in biomineralization of silver by the cell-free extract of P. cyclopium are suggested. Enzymatic synthesis of silver nanoparticles by the mycelium and the cell-free extract of P. cyclopium is excluded.