**5. Effects of photodynamic therapy on** *S. aureus* **biofilms**

*S. aureus* is one of the most important etiologic agents of HAIs, in part, due to the ability of *S. aureus* to form BF. The BF provides a microenvironment that protects bacteria from the immune system's action and antibiotics, providing an extended virulence to the strain [10]. The BF formed by *S. aureus* are communities of microorganisms integrated into a matrix of extracellular polymers. The matrix comprises adhesion polysaccharides and extracellular enzymes, which have shown aggressive behavior [48].

Infections by organisms that produce BF are an important challenge in medical practice, leading to new therapeutic strategies. PDT has been a central focus and

shows mixed results in the literature. Studies using TBO as PS to eradicate *S. aureus* BF have shown a significant reduction in bacterial viability [33, 34, 48]. Noteworthy Anju *et al*. [25], used silica nanoparticles to enhance the antimicrobial efficacy of TBO. The authors evaluated the anti-BF efficacy of the photoactivated TBO silica nanoparticles against *Pseudomonas aeruginosa* (*P. aeruginosa*) as well as *S. aureus.* The results showed that the PDT reduced the viability in *P. aeruginosa* by 66.39 ± 4.22% and the viability of *S. aureus* by 76.22 ± 3.45%. Regarding the controls, the use of TBO alone resulted in an inhibition of 27.28 ± 1.87 and 48.52 ± 1.91% for BF formation by *P. aeruginosa* and *S. aureus*, respectively [25]. A modification in the encapsulation of TBO for PDT was achieved employing carbon nanotubes, which were useful and showed improved results over BF of *P. aeruginosa* and *S. aureus* [33]. The anti-BF activity of TBO with nanotubes after exposure to light was 69.94% and 75.54% for *P. aeruginosa* and *S. aureus*, respectively. Compared to the study by Anju *et al*. [25], the photoinactivation of bacteria was much higher, and cell viability and exopolysaccharide production were more reduced [33].

Authors using indocyanine green (ICG) as PS observed mixed results [29, 31]. For example, Li *et al*. [31] compared the effect of adding EDTA to ICG for PDT on planktonic and BF bacteria. The results showed that PDT induced by ICG -EDTA combination has a more pronounced antibacterial effect in *S. aureus* and *P. aeruginosa* than PDT with ICG alone. In turn, *P. aeruginosa* was more sensitive to ICG -EDTA PDT than *S. aureus.* Also, PDT combined with antibiotic treatment contributed significantly to eradicating bacteria and disrupting the BF structure. Different results were obtained when combining polydopamine nanoparticles with ICG for PDT of orthopedic titanium implants [29]. Evaluations demonstrated that PDT-mediated ROS and nor hyperthermia were sufficient by themselves to achieve a significant bactericidal effect on *S. aureus* BF. However, both effects, local hyperthermia and ROS production, were synergistic and effectively inhibited most *S. aureus* BF [29].

The photodynamic activity of Curcumin (Cur) by high photooxidation was demonstrated to efficiently abolishing *S. aureus* BF [30, 39]. The group of Geraldo *et al*. [30] established the efficacy of Cur -PDT over MSSA and MRSA BF. The results showed that concentrations as low as 20–40 μM resulted in 1log10 reduction of MSSA BF, but the effect reaches 3log10 inactivation at 80 μM. For MRSA BF, it was observed that at 20 μM of Cur produced a reduction of 1log10, and similarly higher concentrations, 40 and 80 μM, decreased the bacterial survival to 2 log10 in a dose-dependent activity [39]. Hypericin (HYP) is one of the natural derivatives widely used in PDT for the elimination of *S. aureus* BF [17, 20]. However, its bactericidal effect is only achieved in combination with N-acetylcysteine (NAC) [20]. The combination of HYP-NAC in PDT is able to interrupt the preformed BF of *S. aureus* (ATCC 25923), reducing the bacterial viability between 5.2 to 6.3 log10. The treatment for clinical isolates demonstrated similar bactericidal activity, decreasing the viability by 5.5–6.7 log10 [20]. Gao *et al*. [49] showed that zinc phthalocyanine (ZnPc) generates ROS during the PDT treatment of *S. aureus* BF. According to his flow cytometric studies, the bacterial DNA was severely damaged [49]. Finally, combining iodine IR780- PDT with thermal phototherapy (PTT) is effective both *in vitro* and *in vivo* [35]. The authors observed that antibacterial treatment applying only PDT or PTT is not effective in completely eradicating already formed BF [35].

### **6. Modulation in gene expression by photodynamic therapy**

Without considering prophages, plasmids, and transposons, the *S. aureus* genome core is a circular chromosome of approximately 2,800 Kb. The genes that encode virulence factors in *S. aureus* may be contained in the core genome and in

accessory elements. Genes encoding virulence factors can be transferred between different staphylococci strains or transferred to bacteria from other species, including Gram-negative bacteria. In *S. aureus*, the expression of virulence genes is controlled by several regulatory genes; the most studied is the *agr* gene (accessory genetic regulator). The *agr* gene has become associated with a quorum-sensing (QS) system. The RNAIII gene is the main effector of the *agr* system. It acts as a small RNA that regulates the expression of many virulence factors, including most of the genes that encode proteins associated with the cell wall and extracellular structures [50]. These factors are also associated with the formation of BF. Given its importance, the *agr* system can be a good therapeutic target for treating acute and chronic infections associated with the formation of BF. [23]. Therefore, the researchers emphasize the use of PDT can interfere with these systems' actions by inhibiting the spread of BF-forming strains [16, 23, 24].

Pourhajibagher *et al*. [50], evaluated in multiple species, including *S. aureus*, the cell viability of bacterial BF subjected to ICG as a photosensitizer. The gene expression of the QS system and the *arg* gene was determined and compared to untreated bacteria. In both *S. aureus* and other bacteria, *agr* gene and QS gene expression levels decreased after PDT. The *agr* gene expression was reduced approximately 3.7 times, as well as the bacterial viability of *S. aureus* decreased between 42 and 82%, revealing an association of the gene with bacterial BF [23]. In another study, Mahmoudi *et al*. [3] determined the ICA operon gene expression changes in bacteria subjected to sub-lethal doses of PDT in clinical isolates from wound infections in patients with burns. The ICA gene regulates *S aureus* BF production. A significant decrease in the expression of the ICA gene was observed in all *S. aureus* isolates after treatment, suggesting that the inactivation of virulence factors through interference in the expression of the ICA gene by PDT may reduce the pathogenesis of *S. aureus* [3].

One of the objectives that PDT seeks is to modulate the virulence of multiresistant strains by repressing the expression levels of genes involved in bacterial resistance. An example was that of Huang *et al*. [37], who studied the expression response of a specific MRSA gene (*nuc* gene). The *nuc* gene encodes the expression of a thermally stable extracellular nuclease produced by *S. aureus*. The authors observed that this gene's transcription, ordinarily high, was down-regulated after PDT, suggesting the treatment interferes with its expression [37].
