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 log(10) (≥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.

Ion-induced stacking of photosensitizer molecules can remarkably affect the luminescence detection of singlet oxygen in Candida albicans cells.

Singlet oxygen (¹O2) is an important reactive intermediate in photodynamic reactions, particularly in antimicrobial PDT (aPDT). The detection of ¹O2 luminescence is frequently used to elucidate the role of ¹O2 in various environments, particularly in microorganisms and human cells. When incubating the fungus, Candida albicans, with porphyrins XF73 (5,15-bis-[4-(3-Trimethylammonio-propyloxy)-phenyl]-porphyrin) or TMPyP (5,10,15,20-Tetrakis(1-methyl-4-pyridinio)-porphyrin tetra(p-toluenesulfonate)), the ¹O2 luminescence signals were excellent for TMPyP. In case of XF73, the signals showed strange rise and decay times. Thus, ¹O2 generation of XF73 was investigated and compared with TMPyP. Absorption spectroscopy of XF73 showed a change in absorption cross section when there was a change in the concentration from 1×10⁻6M to 1×10⁻³ M indicating an aggregation process. The addition of phosphate buffered saline (PBS) substantially changed ¹O2 luminescence in XF73 solution. Detailed experiments provided evidence that the PBS constituents NaCl and KCl caused the change of ¹O2 luminescence. The results also indicate that Cl- ions may cause aggregation of XF73 molecules, which in turn enhances self-quenching of ¹O2 via photosensitizer molecules. These results show that some ions, e.g., those present in cells in vitro or added by PBS, can considerably affect the detection and the interpretation of time-resolved luminescence signals of ¹O2, particularly in in vitro and in vivo. These effects should be considered for any other photosensitizer used in photodynamic processes.

Fungicidal photodynamic effect of a twofold positively charged porphyrin against Candida albicans planktonic cells and biofilms.

BACKGROUND: Antimicrobial photodynamic therapy is an interesting alternative for the treatment of superficial mucocutaneous mycoses. In immunodeficient patients, these infections are frequently recurrent and resistant to the most commonly used antifungal medications. Candida albicans biofilms frequently cause such infections that can even evolve to deep-seated mycoses. MATERIALS & METHODS: The efficiency of a photodynamic therapy was investigated against C. albicans using a twofold positively charged porphyrin (XF-73) in comparison with the well-known fourfold positively charged porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt). RESULTS: After incubation with 0.5 µM of XF-73 for 15 min and irradiation with blue light (12.1 J/cm(2)), the viability of C. albicans planktonic cells decreased by over 6 log10. For biofilm cells, a longer incubation time (4 h) with 1 µM of XF-73 and a light dose of 48.2 J/cm(2) was necessary to achieve over 5 log10 cell killing. Cell killing was mediated by singlet oxygen that was directly detected via its luminescence at 1270 nm in XF-73-incubated C. albicans biofilms for the first time. Antimicrobial photodynamic therapy yielded better results for XF-73 compared with 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine, tetra-p-tosylate salt when using the same conditions. CONCLUSION: This study provides evidence that XF-73 is a highly efficient photosensitizer to kill C. albicans and it would be worthwhile to test this photosensitizer in clinical studies for both prophylaxis and treatment of infections caused by this microorganism, preventing the spread of C. albicans throughout the bloodstream.