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

Dirty hands: photodynamic killing of human pathogens like EHEC, MRSA and Candida within seconds

Hand hygiene is one of the most important interventions for reducing transmission of nosocomial life-threatening microorganisms, like methicillin resistant Staphylococcus aureus (MRSA), enterohemorrhagic Escherichia coli (EHEC) or Candida albicans. All three pathogens have become a leading cause of infections in hospitals. Especially EHEC is causing severe diarrhoea and, in a small percentage of cases, haemolytic-uremic syndrome (HUS) as reported for E. coli 104:H4 in Germany 2011. We revealed the possibility to inactivate very fast and efficiently MRSA, EHEC and C. albicans using the photodynamic approach. MRSA, EHEC and C. albicans were incubated in vitro with different concentrations of TMPyP for 10 s and illuminated with visible light (50 mW cm−2) for 10 and 60 s. 1 μmol l−1 of TMPyP and an applied radiant exposure of 0.5 J cm−2 achieved a photodynamic killing of ≥99.9% of MRSA and EHEC. Incubation with higher concentrations (up to 100 μmol l−1) of TMPyP caused bacteria killing of >5 log10 (≥99.999%) after illumination. Efficient Candida killing (≥99.999%) was achieved first at a higher light dose of 12 J cm−2. Different rise and decay times of singlet oxygen luminescence signals could be detected in Candida cell suspensions for the first time, indicating different oxygen concentrations in the surrounding for the photosensitizer and singlet oxygen, respectively. This confirms that TMPyP is not only found in the water-dominated cell surrounding, but also within the C. albicans cells. Applying a water–ethanol solution of TMPyP on ex vivo porcine skin, fluorescence microscopy of histology showed that the photosensitizer was exclusively localized in the stratum corneum regardless of the incubation time. TMPyP exhibited a fast and very effective killing rate of life-threatening pathogens within a couple of seconds that encourages further testing in an in vivo setting. Being fast and effective, antimicrobial photodynamic applications might become acceptable as a tool for hand hygiene procedures and also in other skin areas

Retrospective genome-oriented analysis reveals low transmission rate of multidrug-resistant Pseudomonas aeruginosa from contaminated toilets at a bone marrow transplant unit

Background Prevention of toilet-to-patient transmission of multidrug-resistant Pseudomonas aeruginosa (MDR PA) poses management-related challenges at many bone marrow transplant units (BMTUs). Aim To conduct a longitudinal retrospective analysis of the toilet-to-patient transmission rate for MDR PA under existing infection control (IC) measures at a BMTU with persistent MDR PA toilet colonization. Methods The local IC bundle comprised: (1) patient education regarding IC; (2) routine patient screening; (3) toilet flushing volume of 9 L; (4) bromination of toilet water tanks, and (5) toilet decontamination using hydrogen peroxide. Toilet water was sampled periodically between 2016 and 2021 (minimum every three months: 26 intervals). Upon MDR PA detection, disinfection and re-sampling were repeated until ≤3 cfu/100 mL was reached. Whole-genome sequencing (WGS) was performed retrospectively on all available MDR PA isolates (90 out of 117 positive environmental samples, 10 out of 14 patients, including nine nosocomial). Findings WGS of patient isolates identified six sequence types (STs), with ST235/CT1352/FIM-1 and ST309/CT3049/no-carbapenemase being predominant (three isolates each). Environmental sampling consistently identified MDR PA ST235 (65.5% ST235/CT1352/FIM-1), showing low genetic diversity (difference of ≤29 alleles by core-genome multi-locus sequence typing (cgMLST)). This indicates that direct toilet-to-patient transmission was infrequent although MDR PA was widespread (detection on 79 occasions, detection in every toilet). Only three MDR PA patient isolates can be attributed to the ST235/CT1352/FIM-1 toilet MRD PA population over six years. Conclusion Stringent targeted toilet disinfection can reduce the potential risk for MDR PA acquisition by patients

Learning from Embryogenesis—A Comparative Expression Analysis in Melanoblast Differentiation and Tumorigenesis Reveals miRNAs Driving Melanoma Development

Malignant melanoma is one of the most dangerous tumor types due to its high metastasis rates and a steadily increasing incidence. During tumorigenesis, the molecular processes of embryonic development, exemplified by epithelial–mesenchymal transition (EMT), are often reactivated. For melanoma development, the exact molecular differences between melanoblasts, melanocytes, and melanoma cells are not completely understood. In this study, we aimed to identify microRNAs (miRNAs) that promote melanoma tumorigenesis and progression, based on an in vitro model of normal human epidermal melanocyte (NHEM) de-differentiation into melanoblast-like cells (MBrCs). Using miRNA-sequencing and differential expression analysis, we demonstrated in this study that a majority of miRNAs have an almost equal expression level in NHEMs and MBrCs but are significantly differentially regulated in primary tumor- and metastasis-derived melanoma cell lines. Further, a target gene analysis of strongly regulated but functionally unknown miRNAs yielded the implication of those miRNAs in many important cellular pathways driving malignancy. We hypothesize that many of the miRNAs discovered in our study are key drivers of melanoma development as they account for the tumorigenic potential that differentiates melanoma cells from proliferating or migrating embryonic cells

Photodynamic inactivation of bacteria to decolonize meticillin-resistant Staphylococcus aureus from human skin

Background To prevent infections that arise from the skin surface it is necessary to decolonize human skin prior to any proposed treatment or surgical intervention. Photodynamic inactivation of bacteria (PIB) uses cationic photosensitizers that attach to the surface of bacteria, generate reactive oxygen species on light irradiation and thereby kill bacteria via oxidative mechanisms. Objectives To evaluate the potential and the safety of PIB for decolonization of bacteria from skin. Methods PIB with the new photosensitizer SAPYR [2-((4-pyridinyl)methyl)-1H-phenalen-1-one chloride] was initially tested against different bacterial species in vitro. Then, ex vivo porcine skin samples were used as a model for decolonization of different bacteria species. The numbers of viable bacteria were quantified and the mitochondrial activity of skin cells was histologically analysed (using nitroblue tetrazolium chloride, NBTC). The same procedure was performed for human skin and meticillin-resistant Staphylococcus aureus (MRSA). Results The in vitro studies showed a 5 log(10) reduction of all tested bacterial species. On ex vivo porcine skin samples, PIB reduced the viability of all tested bacterial species by at least 3 log(10) steps. On human skin samples ex vivo, PIB reduced the number of viable MRSA by maximal 4 center dot 4 log(10) steps (1000 mu mol L-1 SAPYR, incubation time 10 min, 60 J cm(-2)). NBTC staining showed normal mitochondrial activity in skin cells after all PIB modalities. Conclusions The results of this study show that PIB can effectively and safely kill bacteria like MRSA on the skin surface and might have the potential of skin decolonization in vivo