## 3 Results and Discussion
investigated the synergy between photo- and sonodynamic therapy against Gram-positive bacterial biofilm (_Staphylococcus aureus_) with curcumin (80 uM) as a sensitizer. In this study, an aqueous solution of curcumin with 0.5% DMSO was used. The sample containing the bacteria was first sonicated (1 MHz for 32 min) and then irradiated (450 nm, 70 J/cm\({}^{2}\)) [31]. The results were a 1.67 log reduction for SDT, 2.39 for PDT, and 3.48 for SPDT. However, it should be noted that the applied wavelengths of light was outside the optimal range known as the \"therapeutic window\" [32]. PDT's optimal light wavelength range is 600-800 nm [21]. The use of light with a length of less than 600 nm significantly limits the application potential of the method due to the low permeability of this range through human tissues. On the other hand, using wavelengths higher than 800 nm results in the significantly lower efficiency of ROS generation [32]. The current study proposed the use of two popular and well-researched dyes: Ce6 and RB [18]. Both dyes used have numerous advantages. The toxicity of both is well-known and documented. Ce6 is also registered in many countries as a drug utilized in photodynamic therapy against cancer [33]. On the other hand, RB has been used in the past as a contrast agent in liver imaging [19]. In the presented experiment, Ce6 (1 uM), excited with light at the power of 50 J/cm\({}^{2}\), resulted in a reduction in MRSA bacteria at the level of 2.43 log (no dark toxicity, Figure 5). Neither the increase in the light dose to 100 J/cm\({}^{2}\) or the PS concentration to 10 uM resulted in a statistically significant enhancement of the bactericidal effect against MRSA. Ce6 has been tested in PACT against several microorganisms, including _P. acnes, E. coli, C. albicans, S. epidermidis, E. faecalis, E. coli, P. mirabilis, P. aeruginosa, and S. enterica_ so far [34; 35; 36; 37; 38]. Previous research on MRSA performed by [PERSON] et al. and [PERSON] et al. gave confusing results [22; 39]. In the case of the first experiment, a 5 log reduction was achieved, but a dose of \(>\)128 uM was used [22]. In the dark toxicity tests performed here, such a high concentration of PS was bactericidal against the used strain. Research by [PERSON] et al. [39] assessed the bactericidal effect by observing the inhibition zones. This method does not allow for an accurate quantitative assessment of the bactericidal effect. However, irradiation with light with a power of \(<\)50 J/cm\({}^{2}\) gave only the effect described by the authors as \"mild inhibition\" [39]. RB, a SS, at the concentration of 100 and 10 uM, revealed very strong dark toxicity to the tested strain. Therefore, the concentration was reduced to 1 uM RB (no dark toxicity). The SS in such an amount was subjected to ultrasounds at an energy dose of 256 J/cm\({}^{2}\) (40% duty cycle, 3 W, 1 MHz), and a \(<\)1 log reduction was noticed as the SDT result. Increasing the ultrasound energy to 512 J/cm\({}^{2}\) did not significantly change the antibacterial potential of the therapy. The use of SDT with RB as an SS is relatively innovative. Thus far, only one study by [PERSON] et al. has used pure RB as an SS (15 uM, 28 kHz, 0.84 W/cm\({}^{2}\)  
Figure 4: Job’s plot of RB vs. Ce6; (**A**) UV-Vis spectra during the addition of Ce6 equivalents to RB; (**B**) (I\(-\)I\({}_{0}\))/I\({}_{0}\) vs. Ce6 eq during the addition of Ce6 equivalents to RB (concentration of the host—RB— \(2\times 10^{-5}\))—Job’s plot. 100% duty cycle) and reached maximum activity at the level of a 3-4 log reduction in bacterial growth [40]. In the present study, such a high concentration of RB eradicated MRSA without excitation (dark toxicity). The mentioned tendency could be linked with the phenomenon of so-called \"collateral sensitivity\", which makes antibiotic-resistant strains more sensitive to other chemicals, which has been described in the literature so far [16; 41; 42]. It should also be emphasized that the strains used by [PERSON] et al.