Abstract

The bacterium Legionella pneumophila is still one of the probable causes of waterborne diseases, causing serious respiratory illnesses. In the aquatic systems, L. pneumophila exists inside free-living amoebae or can form biofilms. Currently developed disinfection methods are not sufficient for complete eradication of L. pneumophila biofilms in water systems of interest. Photodynamic inactivation (PDI) is a method that results in an antimicrobial effect by using a combination of light and a photosensitizer (PS). In this work, the effect of PDI in waters of natural origin and of different hardness, as a treatment against L. pneumophila biofilm, was investigated. Three cationic tripyridylporphyrins, which were previously described as efficient agents against L. pneumophila alone, were used as PSs. We studied how differences in water hardness affect the PSs’ stability, the production of singlet oxygen, and the PDI activity on L. pneumophila adhesion and biofilm formation and in biofilm destruction. Amphiphilic porphyrin showed a stronger tendency for aggregation in hard and soft water, but its production of singlet oxygen was higher in comparison to tri- and tetracationic hydrophilic porphyrins that were stable in all water samples. All three studied porphyrins were shown to be effective as PDI agents against the adhesion of the L. pneumophila to polystyrene, against biofilm formation, and in the destruction of the formed biofilm, in their micromolar concentrations. However, a higher number of dissolved ions, i.e., water hardness, generally reduced somewhat the PDI activity of all the porphyrins at all tested biofilm growth stages.

Highlights

  • Photodynamic therapy (PDT) is used to treat various oncological and nononcological diseases by generating cytotoxic single oxygen (1 O2 ) and other reactive oxygen species (ROS) at the site of light activation of a photosensitizer (PS) molecule [1]

  • The water sample from the water well in Prelog, with 403 mg/L CaCO3 dissolved, was throughout our studies considered as hard water (HW), while the water sample from the water well in Nedelišće, with 231 mg/L CaCO3 dissolved, was considered as soft water (SW)

  • Our results showed that water hardness does not strongly affect Legionella biofilm or biofilm formation; a small increase proportional to the amount of dissolved minerals in water can be observed both in the samples irradiated with violet light and the samples kept in the dark

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Summary

Introduction

Photodynamic therapy (PDT) is used to treat various oncological and nononcological diseases by generating cytotoxic single oxygen (1 O2 ) and other reactive oxygen species (ROS) at the site of light activation of a photosensitizer (PS) molecule [1]. Antibacterial photodynamic therapy (aPDT), familiar under the name of photodynamic inactivation (PDI), can be used against various pathogens, so far mostly tested on Escherichia coli and Staphylococcus aureus [2,3], and for treating various infections, such as wound infections, acne, and periodontal diseases [4,5]. Biofilms includes the treatments of topical infections [6], oral biofilms in dentistry [7,8], and biofilms on prosthetic material/implants [9]. It is well known from the literature and research so far that all PSs, neutral, anionic, and cationic, can be effective against Gram-positive bacteria; only positively charged. In the case of anionic or neutral PSs, outer membrane disrupting agents, such as ethylenediaminetetraacetic acid and calcium chloride, are necessary to enable their photodynamic action against

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