Photodynamic Inactivation of Bacterial Contaminations: Application to Biofilms

In 1999, it was discovered that bacteria prefer to live within complex communities that are attached to interfaces, called biofilms. Since this discovery, the interest of the scientific community to explore this new bacterial lifestyle aroused. Among the different types of contaminations, bacterial biofilms are considered to be the most difficult to eradicate since the extracellular polymeric substances, self-produced by the bacterial cells, act as a barrier to external agents. Due to the emerging problems related to the growing ability of microorganisms to tolerate common antimicrobial strategies (e.g., chemicals, antibiotics), innovative and unconventional technologies are required today for the fight against microbial contaminations. There is also a growing desire of the industry and consumers towards methods that are fast, easy to apply, characterized by low costs for both maintenance and purchase, safe to use and environmentally friendly. In this context that provides challenges in dealing with bacterial contaminations, an emerging technology that has shown positive outcomes is photodynamic inactivation (PDI). The technology makes use of light within the visible spectrum in the presence of oxygen-containing species to tackle microorganisms. Two different methods of applying PDI are possible: (i) one exploits the photosensitizers (PS) produced by bacteria that are intrinsically present within their cells, i.e., endogenous PSs, (ii) the second requires the external addition of exogenous PSs. Both methods have demonstrated promising results and effectiveness for the inactivation of bacterial contaminations within different sectors such as the food industry and clinical settings. This PhD research aims to determine the impact of the PDI as a decontamination technology for bacteria with special attention towards biofilms. In order to assess this overall goal, three main objectives are achieved. To achieve the first objective, a protocol for obtaining a reproducible biofilm on a flat, wide polystyrene surface was developed for Pseudomonas fluorescens, a Gram-negative bacterium. This process gave insight into the evolution of the biofilms in terms of viable cells and biomass over 24 hours. The protocol was also successfully validated for a Gram-positive bacterium, Staphylococcus epidermidis. To meet the second objective, the impact of the PDI with endogenous PSs was studied on the two bacteria P. fluorescens and S. epidermidis. The aims of this second part are twofold: first, to provide an understanding of PDI applied to different types of bacterial contaminations and, second, to examine the effect of PDI when biofilms are considered. Biofilms can be found both hydrated and not hydrated, the way they are affected by PDI can be different. For this reason both of them were considered for the research in Chapter 5 and 6, respectively, since both can be found on equipment, tools and in industrial water tanks. First, the effect of violet, blue, green and red light-emitting diode irradiation was investigated on three different types of contaminations of P. fluorescens. A short treatment of 30 minutes was tested on the planktonic form, single cells inoculated on a surface, and hydrated biofilms. Among the different colours, violet and blue light were found to inactivate some types of contaminations at different extents. (i) For planktonic cells, violet light slightly inactivated the population. (ii) For individual cells on a surface, violet light caused a consistent reduction below the detection limit, while blue light was effective when salt stress was added to the system. (iii) Hydrated biofilms were not affected by any of the light sources. Second, the effect of five different colours, i.e., violet, blue, green, yellow and red, and three or four different irradiance conditions (2.5, only for violet light, 25.0, 75.0 and 100.0%) were tested on biofilms of P. fluorescens and S. epidermidis. (i) Violet light caused a high reduction of both species, with 6.80 and 3.69 log reductions, respectively, (ii) blue light was only effective against P. fluorescens using harsher conditions (100% of maximum irradiance) and (iii) green, yellow and red irradiation did not have any effect. Finally, to fulfil the third objective, the PDI was investigated using exogenous PSs and adjuvants excited by the means of white light both on P. fluorescens and S. epidermidis. Three surfactants (non-ionic, anionic and cationic) are used as adjuvants for PDI on the Gram-negative P. fluorescens and the Gram-positive S. epidermidis, while chelating agents are used only against P. fluorescens. White light in combination with exogenous PSs promotes a bactericidal effect within 30 minutes of irradiation on S. epidermidis, but not on P. fluorescens. Moreover, among all the adjuvants, some of them enhance the inactivation of S. epidermidis. But none of them enhances the effect on P. fluorescens. In conclusion, this PhD thesis demonstrates that PDI both with endogenous PSs and exogenous PSs is a promising technology that can be used for different types of bacterial contaminations, even against biofilms, which are the most recalcitrant to several kinds of remedies. The findings of this dissertation are encouraging and will serve as a base for future research in the field of PDI.status: publishe