## 2 Results
To facilitate analysis of the obtained results, the degree of death of the isolates was expressed as the average reduction in the number of viable cells (%), which was calculated as the arithmetic means of the mortality level of the strains marked 3374, 3375, 3515, 3690 (Tables S4 and S5). The results obtained showed that the average killing effect of the studied _S. aureus_ isolates was significantly higher compared to MB at a concentration of 31.25 mg L\({}^{-1}\). Figure 3: Effect of MB (Methylene Blue), MB + AuBNPs, and MB+AuChNPs (Methylene Blue + gold nanoparticles mixture) on the viability of biofilm formed on the surface of stainless steel discs by _S. aureus_ 3374 (**a**); _S. aureus_ 3375 (**b**); _S. aureus_ 3515 (**c**); _S. aureus_ 3690 (**d**) after exposure to laser light for 30 min (energy fluence was 189 J cm\({}^{-2}\)). Average \(\pm\)SD of three independent experiments is shown; in all groups, \(p<\) 0.05. Age of biofilm: (24 h) = biofilm formed on the steel surface within 24 h; (48 h) = biofilm formed on the steel surface within 48 h.  
The cell death rate caused by MB, MB + AuBNPs, and MB+AuChNPs averaged 88 \(\pm\) 2, 99.5 \(\pm\) 1.5, and 99.951 \(\pm\) 0.005% for 24-h biofilms formed by the studied strains on the glass discs. LIVE/DEADTM_Bac_LightTM Bacterial Viability Kit (Thermo Fisher Scientific, USA) for monitoring bacterial cell viability as a function of cell membrane integrity was used to evaluate the effectiveness of aPDI against biofilms formed on the glass surface during 24 h of incubation by _S. aureus_ 3515 with MB, MB + AuBNPs and MB+AuChNPs as photosensizers. Dead _occi_ (with damaged cell membranes) were stained red (Figure 4a-d). The increasing amount of red color in the microscopy images corresponded to a higher efficiency of biofilm destruction. Figure 4: _Cont._  
For biofilms growing on a glass surface for 48 h, the photocidal activity of MB, MB+AuBNPs, and MB+AuChNPs averaged 81 \(\pm\) 1.5, 90.8 \(\pm\) 1.3, and 97.98 \(\pm\) 0.95%, respectively (see Table S4; Supplementary Information). Photoeradication of biofilms formed by the MRSA isolates on steel discs in the presence of MB, MB+AuBNPs, and MB+AuChNPs as photosensizers occurred at a similar level (compared to biofilms developed on the glass surface) and the highest level of mortality was observed, reaching 99.948 \(\pm\) 0.021%, when MB+AuChNPs were used as a photosensitizing mixture (Table S5). Another photocidal protocol was performed using MB as photosensitizer at a concentration of 125 mg L\({}^{-1}\), and it was found that this procedure resulted in extremely high efficiency in destroying pathogens. Regardless of strain, type of material surface, or the age of the biofilm, the mortality rate was higher than 99.9% after exposure of the MRSA isolates to the light dose of 189 J cm\({}^{-2}\) (Table S6). The presence of gold nanoparticles in the  
Figure 4: Fluorescence microscopy images of biofilm formed on the glass surface by _S. aureus_ 3515 before aPDI (**a**); after photodynamic inactivation with MB alone as a photosensitizer (**b**); after photodynamic inactivation with MB+AuBNPs as a photosensitizer (**c**); after photodynamic inactivation with MB+AuChNPs as a photosensitizer (**d**). In the presence of the SYTO 9 and propidium iodide mixture, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent orange/red. Subsequent photoelimination of the bacteria consisted of three exposures of the biofilms to laser light in the presence of MB alone at a nontoxic concentration of 31.25 mg L\({}^{-1}\), but in combination with the removal of dead cells (see: _Effect of photodynamic therapy on the viability of the S. aureus biofilm_; Procedure 2). The effectiveness of photodestruction of biofilm in these experimental conditions depended on the isolate, the age of the biofilm, and the type of surface.