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

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

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

Inhibitory effects of calcium or magnesium ions on PDI

Photodynamic inactivation of microorganisms (PDI) finds use in a variety of applications. Several studies report on substances enhancing or inhibiting PDI. In this study, we analyzed the inhibitory potential of ubiquitous salts like CaCl2 and MgCl2 on PDI against Staphylococcus aureus and Pseudomonas aeruginosa cells using five cationic photosensitizers methylene blue, TMPyP, SAPYR, FLASH-02a and FLASH-06a. TMPyP changed its molecular structure when exposed to MgCl2, most likely due to complexation. CaCl2 substantially affected singlet oxygen generation by MB at small concentrations. Elevated concentrations of CaCl2 and MgCl2 impaired PDI up to a total loss of bacterial reduction, whereas CaCl2 is more detrimental for PDI than MgCl2. Binding assays cannot not explain the differences of PDI efficacy. It is assumed that divalent ions tightly bind to bacterial cells hindering close binding of the photosensitizers to the membranes. Consequently, photosensitizer binding might be shifted to outer compartments like teichoic acids in Gram-positives or outer sugar moieties of the LPS in Gram-negatives, attenuating the oxidative damage of susceptible cellular structures. In conclusion, CaCl2 and MgCl2 have an inhibitory potential at different phases in PDI. These effects should be considered when using PDI in an environment that contains such salts like in tap water or different fields of food industry

Photodynamic inactivation – the role of ions and future application perspectives

The careless use of antibiotics led to the emergence of multi-resistant bacteria responsible for 700,000 deaths annually, estimates even predict 50 million annual deaths as of 2050 - especially since new antibiotics are unlikely to be introduced. To counteract this development, new approaches are urgently needed. In both medicine and hygiene, photodynamic inactivation (PDI) can make its contribution in the treatment of human skin or the use of antimicrobial surfaces. In PDI, highly reactive singlet oxygen (¹O₂), responsible for the antimicrobial effect, is formed based on a photosensitizer (PS). PDI shows excellent activity in a controlled laboratory environment, but applications under real live conditions require considerably higher PS concentration or light doses. This hampers the application and requires purposeful research to unfold these obstacles. The aim of this work was to elucidate which substances and processes are involved in this effect to overcome those obstacles, in particular for PDI on human skin. Investigated substances were Na⁺, Ca²⁺ or Mg²⁺ as well as CO₃²⁻ and PO₄³⁻, especially relevant for applications under real life conditions. It was hypothesized, if citrate might circumvent detrimental effects via complexation in artificial solutions containing Ca²⁺ or Mg²⁺ and in tap water or synthetic sweat. The synthetic sweat contained several ions and organic molecules like histidine. Lastly, the results were transferred to the human skin with a PS-hydrogel. Initially, a procedure was established for an efficient investigation of the physical and chemical processes of PDI using SAPYR, a phenalene-based PS as well as the porphyrin TMPyP on Halobacterium salinarum, which requires high NaCl concentrations for growth. It was found that the PS did not change in the high salt environment, still generated ¹O₂, and were able to induce a reduction of viable cells of at least 99.9%. In the next part of this work, it was found that flavin derivatives FLASH-02a and FLASH-06a undergo a chemical reaction with CO₃²⁻ or PO₄³⁻. Consequently, the PS stops generating ¹O₂, accompanied by a quasi-absence of PDI. Similarly, it was shown that regardless of the PS Ca²⁺ and Mg²⁺ have drastic effects on antimicrobial potential. Although the tested PS (methylene blue, TMPyP, SAPYR, FLASH-02a and FLASH-06a) remained chemically intact and generated ¹O₂ a reduced efficacy was demonstrated. Based on this, citrate as chelator was used to circumvent the negative influence of Ca²⁺ and Mg²⁺ and at least for Gram- negative bacteria efficacy was improved. In this context, experiments were also carried out in tap water and synthetic sweat. It was observed that especially histidine in sweat is primarily responsible for an inhibitory effect on PDI. As a final step, the knowledge about inhibitory substances in PDI was used to develop a SAPYR-hydroxyethyl cellulose hydrogel. The hydrogel also contained citrate to circumvent the detrimental effects of Ca²⁺ and Mg²⁺. In all cases, bacterial reduction on human skin was greater than or close to 99.99%. Apoptosis and necrosis did not occur in the skin tissue despite treatment with the PS-hydrogel. The results of this work are of great relevance for the transfer of PDI from laboratory to applications under real life conditions. The data obtained can be used to adapt the application of PDI in Ca²⁺ and Mg²⁺-rich environments like tap water and skin. In the field of dentistry, where PDI is already used, it is advisable to possibly add a washing step with citrate solution to existing protocols to increase effectiveness. The PS-hydrogel could have the greatest contribution to solving the problems mentioned above with multidrug-resistant germs, since it has been shown that good efficacy can be achieved on the skin under acceptable treatment conditions. Finally, many currently used antibiotics or antiseptics in the treatment or prevention of SSTIs already show resistances to the substances used. However, PDI is unlikely to provoke resistance in bacteria because the generated ¹O₂ damages almost all biomolecules via oxidative mechanisms. In this way, the PS-hydrogel described in this thesis may possibly provide a partial answer to the questions of the post-antibiotic age that is upon us

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

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.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Peer Reviewe