Antimicrobial Photoinactivation Research Articles

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell’s susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Novel Broad-Spectrum Antimicrobial Photoinactivation of In Situ Oral Biofilms by Visible Light plus Water-Filtered Infrared A.

Antimicrobial photodynamic therapy (APDT) has gained increased attention as an alternative treatment approach in various medical fields. However, the effect of APDT using visible light plus water-filtered infrared A (VIS + wIRA) on oral biofilms remains unexplored. For this purpose, initial and mature oral biofilms were obtained in situ; six healthy subjects wore individual upper jaw acrylic devices with bovine enamel slabs attached to their proximal sites for 2 h or 3 days. The biofilms were incubated with 100 μg ml(-1) toluidine blue O (TB) or chlorin e6 (Ce6) and irradiated with VIS + wIRA with an energy density of 200 mW cm(-2) for 5 min. After cultivation, the CFU of half of the treated biofilm samples were quantified, whereas following live/dead staining, the other half of the samples were monitored by confocal laser scanning microscopy (CLSM). TB- and Ce6-mediated APDT yielded a significant decrease of up to 3.8 and 5.7 log10 CFU for initial and mature oral biofilms, respectively. Quantification of the stained photoinactivated microorganisms confirmed these results. Overall, CLSM revealed the diffusion of the tested photosensitizers into the deepest biofilm layers after exposure to APDT. In particular, Ce6-aided APDT presented elevated permeability and higher effectiveness in eradicating 89.62% of biofilm bacteria compared to TB-aided APDT (82.25%) after 3 days. In conclusion, antimicrobial photoinactivation using VIS + wIRA proved highly potent in eradicating oral biofilms. Since APDT excludes the development of microbial resistance, it could supplement the pharmaceutical treatment of periodontitis or peri-implantitis.

In Vitro antimicrobial photoinactivation with methylene blue in different microorganisms

AIM: To evaluate the in vitro antimicrobial effects of photodynamic therapy (PDT). METHODS: The microorganism indicators were: Candida albicans, Pseudomonas aeruginosa,Enterococcus faecalis and Staphylococcus aureus. A microbial pool was prepared (108 cells/mL), from which aliquots were transferred to culture plates for carrying out the PDT using methylene blue (50 µM) and low-power laser (660 nm, 100 mW and 9 J).The effect of methylene blue alone, low power laser and the absence of treatments were evaluated. Then, aliquots of 1 µL were plated in a media culture, the number of colony forming units (CFU/mL) was obtained and the data submitted to the F test (ANOVA) with Tamhane's comparisons. RESULTS: The laser radiation in the presence of methylene blue was able to eliminate 74.90% of C. albicans, 72.41% of P. aeruginosa, 96.44% of E. faecalis and 95.42% of S. aureus, thus statistically significant differences were found among the groups (p<0.001). CONCLUSIONS: PDT was effective in reducing the number of viable cells in the studiedmicroorganisms, especially E. faecalis and S. aureus.

Synthesis of decacationic [60]fullerene decaiodides giving photoinduced production of superoxide radicals and effective PDT-mediation on antimicrobial photoinactivation

We report a novel class of highly water-soluble decacationic methano[60]fullerene decaiodides C60[>M(C3N6+C3)2]–(I−)10 [1-(I−)10] capable of co-producing singlet oxygen (Type-II) and highly reactive hydroxyl radicals, formed from superoxide radicals in Type-I photosensitizing reactions, upon illumination at both UVA and white light wavelengths. The O2−·-production efficiency of 1-(I−)10 was confirmed by using an O2−·-reactive bis(2,4-dinitrobenzenesulfonyl)tetrafluorofluorescein probe and correlated to the photoinduced electron-transfer event going from iodide anions to 3C60*[>M(C3N6+C3)2] leading to C60−·[>M(C3N6+C3)2]. Incorporation of a defined number (ten) of quaternary ammonium cationic charges per C60 in 1 was aimed to enhance its ability to target pathogenic Gram-positive and Gram-negative bacterial cells. We used the well-characterized malonato[60]fullerene diester monoadduct C60[>M(t-Bu)2] as the starting fullerene derivative to provide a better synthetic route to C60[>M(C3N6+C3)2] via transesterification reaction under trifluoroacetic acid catalyzed conditions. These compounds may be used as effective photosensitizers and nano-PDT drugs for photoinactivation of pathogens.

Photo‐inactivation of Bacillus endospores: inter‐specific variability of inactivation efficiency

The aims of this work were to (a) evaluate the susceptibility of endospores of Bacillus cereus, B. licheniformis, B. sphaericus and B. subtilis to photodynamic inactivation using a tricationic porphyrin as photosensitizer, (b) assess the efficiency of adsorption of the photosensitizer in endospore material as a determinant of the susceptibility of endospores of different Bacillus species to photo-inactivation, (c) determine the value of B. cereus as a model organism for studies of antimicrobial photodynamic inactivation of bacterial endospores. The results of irradiation experiments with endospores of four species of Bacillus showed that B. cereus was the only species for which efficient endospore photo-inactivation (> 3 log reduction) could be achieved. Endospores of B. licheniformis, B. sphaericus and B. subtilis were virtually resistant to photo-inactivation with tricationic porphyrin. The amount of porphyrin bound to endospore material was not significantly different between species, regardless of the presence of an exosporium or exosporium-like outer layer. The sensitivity of endospores to photodynamic inactivation with a tricationic porphyrin is highly variable among different species of the genus Bacillus. The presence of an exosporium in endospores of B. cereus and B. sphaericus, or an exosporium-like glycoprotein layer in endospores of B. subtilis, did not affect the amount of bound photosensitizer and did not explain the inter-species variability in susceptibility to photodynamic inactivation. The results imply that the use of B. cereus as a more amenable surrogate of the exosporium-producing B. anthracis must be carefully considered when testing new photosensitizers for their antimicrobial photo-inactivation properties.

79 Paradoxical potentiation of antimicrobial photoinactivation by sodium azide using seven phenothiazinium photosensitizers

79 Paradoxical potentiation of antimicrobial photoinactivation by sodium azide using seven phenothiazinium photosensitizers

Strategies to Potentiate Antimicrobial Photoinactivation by Overcoming Resistant Phenotypes†

Conventional antimicrobial strategies have become increasingly ineffective due to the emergence of multidrug resistance among pathogenic microorganisms. The need to overcome these deficiencies has triggered the exploration of alternative treatments and unconventional approaches towards controlling microbial infections. Photodynamic therapy (PDT) was originally established as an anticancer modality and is currently used in the treatment of age-related macular degeneration. The concept of photodynamic inactivation requires cell exposure to light energy, typically wavelengths in the visible region that causes the excitation of photosensitizer molecules either exogenous or endogenous, which results in the production of reactive oxygen species (ROS). ROS produce cell inactivation and death through modification of intracellular components. The versatile characteristics of PDT prompted its investigation as an anti-infective discovery platform. Advances in understanding of microbial physiology have shed light on a series of pathways, and phenotypes that serve as putative targets for antimicrobial drug discovery. Investigations of these phenotypic elements in concert with PDT have been reported focused on multidrug efflux systems, biofilms, virulence and pathogenesis determinants. In many instances the results are promising but only preliminary and require further investigation. This review discusses the different antimicrobial PDT strategies and highlights the need for highly informative and comprehensive discovery approaches.