Photodynamic inactivation of different pathogenic bacteria on human skin using a novel photosensitizer hydrogel.

BACKGROUND: The colonization of skin with pathogenic, partially antibiotic-resistant bacteria is frequently a severe problem in dermatological therapies. For instance, skin colonization with Staphylococcus aureus is even a disease-promoting factor in atopic dermatitis. The photodynamic inactivation (PDI) of bacteria could be a new antibacterial procedure. Upon irradiation with visible light, a special photosensitizer exclusively generates singlet oxygen. This reactive oxygen species kills bacteria via oxidation independent of species or strain and their antibiotic resistance profile causing no bacterial resistance on its part. OBJECTIVE: To investigate the antibacterial potential of a photosensitizer, formulated in a new hydrogel, on human skin ex vivo. METHODS: The photochemical stability of the photosensitizer and its ability to generate singlet oxygen in the hydrogel was studied. Antimicrobial efficacy of this hydrogel was tested step by step, firstly on inanimate surfaces and then on human skin ex vivo against S. aureus and Pseudomonas aeruginosa using standard colony counting. NBTC staining and TUNEL assays were performed on skin biopsies to investigate potential necrosis and apoptosis effects in skin cells possibly caused by PDI. RESULTS: None of the hydrogel components affected the photochemical stability and the life time of singlet oxygen. On inanimate surfaces as well as on the human skin, the number of viable bacteria was reduced by up to 4.8 log10 being more effective than most other antibacterial topical agents. Histology and assays showed that PDI against bacteria on the skin surface caused no harmful effects on the underlying skin cells. CONCLUSION: Photodynamic inactivation hydrogel proved to be effective for decolonization of human skin including the potential to act against superficial skin infections. Being a water-based formulation, the hydrogel should be also suitable for the mucosa. The results of the present ex vivo study form a good basis for conducting clinical studies in vivo.

Photodynamic inactivation of Salmonella enterica and Listeria monocytogenes inoculated onto stainless steel or polyurethane surfaces.

The photodynamic inactivation (PDI) uses molecules (photosensitizers) that absorb visible light (385-450 nm) energy, transfer it to adjacent molecular oxygen and thereby generating the biocidal singlet oxygen and other reactive oxygen species in situ. Efficacy of PDI was tested against Listeria monocytogenes and Salmonella enterica in three ways. Firstly, by adding the photosensitizer to bacterial suspensions. Secondly, bacteria were placed on inanimate surfaces and then sprayed with a photosensitizer suspension. Thirdly, bacteria were placed on coated inanimate surfaces, where the photosensitizer was permanently fixed in this coating (antimicrobial coating, AMC). Experiments were performed without and with soiling (albumin, sheep erythrocytes). In suspension, PDI reduced the number of viable Listeria monocytogenes and Salmonella enterica by more than 6 Log CFU/mL within seconds of light exposure. Photosensitizer spray suspension reduced the bacterial burden on surfaces with up to about 6 Log CFU/mL (5 s light exposure). PDI, even in the presence of high soiling, achieved a reduction of up to 5.1 ± 1.2 Log CFU/mL. The AMC showed a bacterial reduction that decreased from 5.1 to 0.7 Log CFU/mL with increasing soiling. Depending on the soiling and the respective bacteria, the spray suspension or AMC achieved a bacterial reduction on the running conveyor belt demonstrator ranging from 2.9 to 5.3 or 0.5 to 4.5 Log CFU/mL, respectively. PDI used visible light, phenalene-1-one and curcumin photosensitizers, and oxygen from ambient air to reduce the bioburden on typical surfaces in food processing. The AMC acts slower than the spray suspension but enables a permanent, self-sanitizing effect.

Antimicrobial coatings for environmental surfaces in hospitals: a potential new pillar for prevention strategies in hygiene.

Recent reports provide evidence that contaminated healthcare environments represent major sources for the acquisition and transmission of pathogens. Antimicrobial coatings (AMC) may permanently and autonomously reduce the contamination of such environmental surfaces complementing standard hygiene procedures. This review provides an overview of the current status of AMC and the demands to enable a rational application of AMC in health care settings. Firstly, a suitable laboratory test norm is required that adequately quantifies the efficacy of AMC. In particular, the frequently used wet testing (e.g. ISO 22196) must be replaced by testing under realistic, dry surface conditions. Secondly, field studies should be mandatory to provide evidence for antimicrobial efficacy under real-life conditions. The antimicrobial efficacy should be correlated to the rate of nosocomial transmission at least. Thirdly, the respective AMC technology should not add additional bacterial resistance development induced by the biocidal agents and co- or cross-resistance with antibiotic substances. Lastly, the biocidal substances used in AMC should be safe for humans and the environment. These measures should help to achieve a broader acceptance for AMC in healthcare settings and beyond. Technologies like the photodynamic approach already fulfil most of these AMC requirements.

Photodynamic Inactivation of Bacteria in Ionic Environments Using the Photosensitizer SAPYR and the Chelator Citrate†.

Many studies show that photodynamic inactivation (PDI) is a powerful tool for the fight against pathogenic, multiresistant bacteria and the closing of hygiene gaps. However, PDI studies have been frequently performed under standardized in vitro conditions comprising artificial laboratory settings. Under real-life conditions, however, PDI encounters substances like ions, proteins, amino acids and fatty acids, potentially hampering the efficacy of PDI to an unpredictable extent. Thus, we investigated PDI with the phenalene-1-one-based photosensitizer SAPYR against Escherichia coli and Staphylococcus aureus in the presence of calcium or magnesium ions, which are ubiquitous in potential fields of PDI applications like in tap water or on tissue surfaces. The addition of citrate should elucidate the potential as a chelator. The results indicate that PDI is clearly affected by such ubiquitous ions depending on its concentration and the type of bacteria. The application of citrate enhanced PDI, especially for Gram-negative bacteria at certain ionic concentrations (e.g. CaCl2 or MgCl2 : 7.5 to 75 mmol L-1 ). Citrate also improved PDI efficacy in tap water (especially for Gram-negative bacteria) and synthetic sweat solution (especially for Gram-positive bacteria). In conclusion, the use of chelating agents like citrate may facilitate the application of PDI under real-life conditions.

Antimicrobial Photodynamic Coatings Reduce the Microbial Burden on Environmental Surfaces in Public Transportation-A Field Study in Buses.

Millions of people use public transportation daily worldwide and frequently touch surfaces, thereby producing a reservoir of microorganisms on surfaces increasing the risk of transmission. Constant occupation makes sufficient cleaning difficult to achieve. Thus, an autonomous, permanent, antimicrobial coating (AMC) could keep down the microbial burden on such surfaces. A photodynamic AMC was applied to frequently touched surfaces in buses. The microbial burden (colony forming units, cfu) was determined weekly and compared to equivalent surfaces in buses without AMC (references). The microbial burden ranged from 0-209 cfu/cm2 on references and from 0-54 cfu/cm2 on AMC. The means were 13.4 ± 29.6 cfu/cm2 on references and 4.5 ± 8.4 cfu/cm2 on AMC (p < 0.001). The difference in microbial burden on AMC and references was almost constant throughout the study. Considering a hygiene benchmark of 5 cfu/cm2, the data yield an absolute risk reduction of 22.6% and a relative risk reduction of 50.7%. In conclusion, photodynamic AMC kept down the microbial burden, reducing the risk of transmission of microorganisms. AMC permanently and autonomously contributes to hygienic conditions on surfaces in public transportation. Photodynamic AMC therefore are suitable for reducing the microbial load and closing hygiene gaps in public transportation.

Interplay of phosphate and carbonate ions with flavin photosensitizers in photodynamic inactivation of bacteria.

Photodynamic inactivation (PDI) of pathogenic bacteria is a promising technology in different applications. Thereby, a photosensitizer (PS) absorbs visible light and transfers the energy to oxygen yielding reactive oxygen species (ROS). The produced ROS are then capable of killing microorganisms via oxidative damage of cellular constituents. Among other PS, some flavins are capable of producing ROS and cationic flavins are already successfully applied in PDI. When PDI is used for example on tap water, PS like flavins will encounter various ions and other small organic molecules which might hamper the efficacy of PDI. Thus, the impact of carbonate and phosphate ions on PDI using two different cationic flavins (FLASH-02a, FLASH-06a) was investigated using Staphylococcus aureus and Pseudomonas aeruginosa as model organisms. Both were inactivated in vitro at a low light exposure of 0.72 J cm-2. Upon irradiation, FLASH-02a reacts to single substances in the presence of carbonate or phosphate, whereas the photochemical reaction for FLASH-06a was more unspecific. DPBF-assays indicated that carbonate and phosphate ions decreased the generation of singlet oxygen of both flavins. Both microorganisms could be easily inactivated by at least one PS with up to 6 log10 steps of cell counts in low ion concentrations. Using the constant radiation exposure of 0.72 J cm-2, the inactivation efficacy decreased somewhat at medium ion concentrations but reached almost zero for high ion concentrations. Depending on the application of PDI, the presence of carbonate and phosphate ions is unavoidable. Only upon light irradiation such ions may attack the PS molecule and reduce the efficacy of PDI. Our results indicate concentrations for carbonate and phosphate, in which PDI can still lead to efficient reduction of bacterial cells when using flavin based PS.

First Report on Photodynamic Inactivation of Archaea Including a Novel Method for High-Throughput Reduction Measurement.

Archaea are considered third, independent domain of living organisms besides eukaryotic and bacterial cells. To date, no report is available of photodynamic inactivation (PDI) of any archaeal cells. Two commercially available photosensitizers (SAPYR and TMPyP) were used to investigate photodynamic inactivation of Halobacterium salinarum. In addition, a novel high-throughput method was tested to evaluate microbial reduction in vitro. Due to the high salt content of the culture medium, the physical and chemical properties of photosensitizers were analyzed via spectroscopy and fluorescence-based DPBF assays. Attachment or uptake of photosensitizers to or in archaeal cells was investigated. The photodynamic inactivation of Halobacterium salinarum was evaluated via growth curve method allowing a high throughput of samples. The presented results indicate that the photodynamic mechanisms are working even in high salt environments. Either photosensitizer inactivated the archaeal cells with a reduction of 99.9% at least. The growth curves provided a fast and precise measurement of cell viability. The results show for the first time that PDI can kill not only bacterial cells but also robust archaea. The novel method for generating high-throughput growth curves provides benefits for future research regarding antimicrobial substances in general.

A Closer Look at Dark Toxicity of the Photosensitizer TMPyP in Bacteria.

Photodynamic inactivation of bacteria (PIB) is based on photosensitizers which absorb light and generate reactive oxygen species (ROS), killing cells via oxidation. PIB is evaluated by comparing viability with and without irradiation, where reduction of viability in the presence of the photosensitizer without irradiation is considered as dark toxicity. This effect is controversially discussed for photosensitizers like TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluensulfonate). TMPyP shows a high absorption coefficient for blue light and a high yield of ROS production, especially singlet oxygen. Escherichia coli and Bacillus atrophaeus were incubated with TMPyP and irradiated with different light sources at low radiant exposures (µW per cm²), reflecting laboratory conditions of dark toxicity evaluation. Inactivation of E. coli occurs for blue light, while no effect was detectable for wavelengths >450 nm. Being more susceptible toward PIB, growth of B. atrophaeus is even reduced for light with emission >450 nm. Decreasing the light intensities to nW per cm² for B. atrophaeus, application of TMPyP still caused bacterial killing. Toxic effects of TMPyP disappeared after addition of histidine, quenching residual ROS. Our experiments demonstrate that the evaluation of dark toxicity of a powerful photosensitizer like TMPyP requires low light intensities and if necessary additional application of substances quenching any residual ROS.