## 3 Discussion
Furthermore, young biofilms were more sensitive to photodestruction compared to older structures, and these observations are consistent with the results presented by [PERSON] et al. [22]. Furthermore, it seems that more intense biofilm growth was on the surface of glass discs, and these cultures were more sensitive to laser light photosensitization. It has already been suggested that the efficiency of biofilm formation (as well as its removal) may be related to the surface nature [22; 23]. Cell adhesion to the surface is one of the main factors that influence the formation of biofilms in various materials [24; 25; 26]. The use of MB as a photosensitizer at a concentration of 62.5 mg L\({}^{-1}\) has been shown to allow the protocol to be accepted as compliant with the requirements of the American Microbiological Society (AMS), but only for young biofilms in the presence of AuChNPs in the photosensitizer mixture (at least 99.9% cell death is required for the procedure to be considered antibacterial [22; 27]) (see Tables S4 and S5). Chemically synthesized gold nanoparticles appeared to be a slightly better adjuvant. Due to the unsatisfactory results of biofilm removal described above, in the next part of our experiments, aPDT was carried out using MB at a concentration of 125 mg L\({}^{-1}\). It should be emphasized that the extremely high photoeradication efficiency of the MRSA pathogens (without the addition of gold nanoparticles) was observed, and this protocol met the requirements of the AMS. The presence of gold nanostructures in the photosensitizing mixture results in a lethal effect (the number of bacteria was below the detection level). In the case of biogenic AuBNPs, a possible mechanism to increase the photobactericidal activity of MB may be the rapid photobleaching of this dye [28]. The effect of AuChNPs on MB photobleaching was also studied, but the obtained results did not clearly confirm this phenomenon, therefore, it was hypothesized that the interaction of MB and gold nanoparticles during photodynamic cell destruction may result in the more effective production of toxic oxidative species. It is known that in photosensitization, MB may have two different mechanisms of action [29]. The excitation of MB by light can induce the singlet and triplet stages of the molecule and transfer energy through electrons (type I mechanism) or energy (type II mechanism). The Type I mechanism can lead to the formation of superoxide anions (O\({}_{2}\)\({}^{\bullet-}\)), hydroxyl radicals (HO\({}^{\bullet}\)), and hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)). The Type II mechanism proceeds through singlet oxygen, resulting mainly in the breakage of nucleic acids, mostly at the guanosine site. Most authors suggested that \({}^{1}\)O\({}_{2}\) is the major destructive factor in PDT in vivo (this anion is a short-lived radical) [30]. However, it should be emphasized that both type I and type II mechanisms may occur simultaneously, leading to comparable oxidative cell damage, and the ratio of these pathways depends on the cellular microenvironment in which MB accumulates, including the concentration of molecular oxygen [14; 31]. Moreover, there may be differences in the mode of action of aPDI in biofilms compared to bacteria cultivated in planktonic cultures due to extracellular polymeric substances and a potentially reduced penetration depth throughout the biofilms [14]. Detection of harmful ROS and singlet oxygen that could be generated by MB alone and the mixture of MB+gold nanoparticles was performed using common methods (see Section 4.7). There was no significant increase in the production of superoxide anions, hydrogen peroxide, and singlet oxygen in the presence of MB and gold nanoparticles in comparison to MB alone. Statistically significant differences in the level of hydroxyl radicals were only observed, but the obtained results were not unambiguous and depended on the MRSA isolate. For this reason, a fluorimetric assay with dichlorodihydrofluorescein diacetate was used to directly measure the redox state of the cell [32].