*Photodynamic Treatment of* Staphylococcus aureus *Infections DOI: http://dx.doi.org/10.5772/intechopen.95455*

The second most prominent PS is Toluidine Blue (TBO), a hydrophilic cationic PS of phenothiazine dyes with a high 1 O2 quantum yield and strong absorption bands in the 620–660 nm region. Also, it has a high affinity for bacterial membranes and has been approved for clinical use in PDT, and is considered an effective and membranedamaging PS [6]. In all studies where TBO act as a PS, irradiation with red LED light in the range of 630–635 nm was used. This combination increased the antibacterial efficacy of PDT and significantly reduced bacterial viability [3, 6, 24, 25, 33, 34]. One of the most prominent studies was carried out by the group of Zhyhyu Cai et al. [19], whose objective was to evaluate how effective the disinfection by combining antiseptics with PDT is in *S. aureus* BF present on the titanium surface is. The results indicate that the administration of antiseptics such as chlorhexidine or H2O2 with PDT was the most effective protocol, producing a reduction of approximately 3–4 log10 in the number of adhering bacteria compared to any treatment alone. In addition to bacterial reduction, it was the first study *in vitro* to evaluate the antibacterial effects of the concurrent application of antiseptics with PDT against *S. aureus* BF presented on the surface of Titanium [34].

Several natural PS derived from plants are also highlighted to be used for PDT, such as the sinoporphyrin sodium (S-PS). For example, Mai *et al*. [28], and Jia *et al*. [4], used a similar methodology for activation of S-PS, employing red LED light irradiation. The results indicate that PDT therapy with S-PS has significant antibacterial activity on MDSSA and MDRSA bacteria [4, 28]. Also, the S-PS exceeds the solubility of other PS in a physiological environment and proves to be an ideal PS with low dark toxicity, as well as having high purity and easy extraction. Another natural PS widely used in *in vitro* and *in vivo* studies is hypericin (HYP), polycyclic quinine extracted from *Hypericum perforatum* (commonly known as St. John's wort) and derives its name from the plant. Previous interest in this herb has focused on its antidepressant effects. This plant has been evaluated and tested for its photooxidative activity against a series of bacterial and fungal strains in recent years. It has several desirable properties as PS, including a high 1 O2 generation quantum yield, a high extinction coefficient close to 600 nm, and relatively low dark toxicity. In studies carried out by the group of Nafee *et al*. [17], the quantity as low as 0.03 M of HYP inhibited *in vitro* 60–120% the growth of BF producing MRSA strains compared to planktonic cells, 55–75%. *In vivo* studies on rats showed higher wound healing potential, better epithelialization, and keratinization of the skin in infected wounds treated with HYP nanoparticles [17]. Finally, hypocrellin B, an active component of traditional Chinese medicine from the herb *Hypocrella bambuase*, was tested for PDT. Numerous studies have shown its antiviral, antibacterial, and antifungal effects and antitumor activity. Interestingly, this PS is also a strong ROS generator when activated by visible light. Yuan Jiang *et al*. [27] observed a significant decrease in the viability of *S. aureus* after LED light irradiation. Remarkable ultrastructural damage was also evidenced in bacterial cells due to the photodynamic action of hypocrellin B [18].

### **3. Photodynamic therapy in clinical isolates strains**

Most of the studies on PDT for *S. aureus* infections found in the literature come from standard bacterial strains acquired from different microbiological laboratories for *in vitro*, *in vivo,* and *ex vivo* studies. The most used certified reference standard bacterial strains came from the American Type Culture Collection (ATCC). ATCC strains are certified microorganisms for quality control in microbiology. Also, their genotypic and phenotypic characteristics guarantee the identity of the microorganism, and by having this documentation, the laboratory will avoid carrying out


**Table 2.**

*Diversity of the clinical isolates.*

**121**

additional tests for the identification of the strains, which translates into saving time and resources. However, studies in which clinical isolates of *S. aureus* were used to demonstrate the PDT may be useful for bacteria from active infections.

PDT is an approach that has shown promise in treating skin and soft tissue infections, one of the most recent studies of Mahmoudi *et al*. [3], used clinical isolates of *S. aureus* from samples of burn wounds of 95 patients with symptomatic infection. The viability of *S. aureus* isolates was significantly reduced to 40% after 30 sec of exposure to a LED light, with minimal risk of development of resistance [3]. Another study developed by Tawfik *et al*. [21] was carried out over clinal isolates of *S. aureus* from a population of twenty children aged 3 to 5 years diagnosed with impetigo. The PDT was compared for light-irradiated gold, methylene blue (MB), and gold -MB conjugate nanoparticles. It was shown that the maximum inhibitory effect on *S. aureus* was obtained with the gold nanoparticle-MB conjugate [21]. The 5-ALA has also been used for PDT over clinical isolates of BF of MSSA and MRSA strains of *S. aureus* [41]. The results over isolates from samples of adult patients with chronic rhinosinusitis with or without nasal polyps showed a robust bactericidal effect that increased when the PDT was combined with antibiotics treatment [41]. Finally, one of the most relevant studies carried out by Winkler *et al*. [22] tested the effectiveness of the chlorophyll derivative (Ce6) combined with a red light to eradicate *in vitro* a diverse set of clinical isolates of MSSA and MRSA. Those bacterial isolates, their biological sample, origin, and their susceptibility profiles are listed in **Table 2**, showing the diversity of the strains. The *in vitro* study demonstrated that all clinical isolates of MSSA and MRSA were inactivated by PDT when bacterial cells were previously incubated with ≥128 μM Ce6 [22].

### **4. Synergism with antibiotics or other drugs**

Although PDT presents positive expectations for the treatment of MDRSA, several researchers have wanted to anticipate the generation of resistance, and they began the search for an antimicrobial strategy that generates greater potency and better results. Therefore, a new research sub-field has been opened, combining PDT with antibiotic treatment in *S. aureus*.

Gentamicin (GEN) is one of the most widely used antibiotics for treating various HAIs. The GEN is an aminoglycoside, which inhibits protein synthesis binding to the 30S subunit of the bacterial ribosome and causes protein mistranslation and bacterial death. GEN is a broad-spectrum antibiotic used for clinical treatment, although its frequent use has generated a high resistance level. Several authors have evaluated the synergy of combining the GEN with PDT for the antibacterial treatment of *S. aureus*. Most of these studies use a similar methodology, consisting of pre-treatment of the bacteria in the dark with different tested PS concentrations. This is followed by the transfer of treated bacteria in suspensions to microtiter plates containing different GEN concentrations and irradiated with different doses of light. Finally, the bacterial plaque count is performed in the dark to calculate bacterial viability. In this way, a diminution in the GEN-MIC when combined with PDT is determined [6, 19, 42–44]. One of the most representative and updated studies, developed by the group of Liu *et al*. [6], verified the synergistic effects of PDT by combining TBO with GEN. This combination has a better antibacterial activity on MDSSA compared to MDRSA bacteria. The authors observed a dose-dependent effect with a maximum of 9 μg / mL GEN decreased of up to 1.8 log10 the survival of MDSSA bacteria. However, no bactericidal effect was observed on MDRSA at a GEN concentration of up to 150 μg/mL. Suggesting the MDSSA strains are more sensitive to PDT-GEN therapy than MDRSA [6].

#### *Photodynamic Treatment of* Staphylococcus aureus *Infections DOI: http://dx.doi.org/10.5772/intechopen.95455*

Another widely used antibiotic to treat infections caused by multidrug-resistant bacteria is Linezolid (LN). Linezolid is a bacteriostatic antibiotic that binds to bacterial ribosomal RNA, inhibiting protein translation of Gram-positive bacteria. A large body of evidence shows that PDT significantly increases the effectiveness of LN treatment synergistically for different strains of *S. aureus* [26, 27]. Special mention deserves the study by Kashef *et al*. [31], who observed that the combination of TBO and MB in PDT with LN is useful in eradicating *S. aureus* BF in chronic diabetic foot ulcers. By itself, PDT therapy with MB or TBO did not decrease the bacterial viability of any of the *S. aureus* tested strains. However, the combination of MB-PDT and antibiotics resulted in a 1.2 log10 reduction in viability comparing to the antibiotic treatment alone (0.6 log10 reductions) [31]. The ciprofloxacin (CIP) is an antibiotic agent of the quinolone family, which binds to the DNA gyrase-DNA complex. The DNA gyrase allows the DNA to unwind and rotate freely within the cell; thus, CIP produces bacterial death. PDT studies showed synergism with CIP [45, 46]. For example, Ronqui *et al*. [46] observed a significant antibacterial increase, reducing bacterial survival 5.4 log10 in *S. aureus* BF and approximately 7 log10 for *Escherichia coli* BF combining PDT treatment with MB and CIP [46].

One of the most explored resistance mechanisms in the last two decades has been that of vancomycin-resistant *S. aureus* (VRSA). VAN acts on the synthesis of the bacterial cell-wall, inhibiting the formation of peptidoglycans. Not only the permeability of the cytoplasmic membrane but also the RNA synthesis is altered. Strains of VRSA can transfer their resistance mechanism to other pathogenic bacteria such as *Enterococcus faecalis,* causing a significant number of HAIs outbreaks [47]. For this reason, PDT has been studied as a therapeutic option that enhances the bactericidal action of VAN [10, 41]. Di poto et al. [10], demonstrated that pre-treatment of clinical MSSA and MRSA strains producers of BF with PDT, followed by the addition of NPV, causes disruption of the BF matrix and allows complete elimination of the bacteria. Synergism with PDT improved the antimicrobial activity of VAN with a five-fold decrease in bacterial viability compared to samples treated with PDT alone [10].

The synergism of PDT has also been explored with other drugs such as mucolytics, anticoagulants, antiseptics, and disinfectants. In general, these studies present encouraging results. All showed decreased bacterial viability in combined therapy with these different compounds [11, 20, 31, 34, 35]. For example, Braz *et al*. [11] showed both *in vitro* and *ex vivo* the efficacy of PDT mediated by a formulation based on a non-separate mixture of cationic porphyrins (FORM) in combination with potassium iodide (KI) or povidone-iodine (PVP-I) for photoinactivation of MRSA in the skin. The in vitro results demonstrated that the FORM + KI combination was an effective therapy in a dose-depended manner. Results *ex vivo*, shown a reduction of 3.1 Log10 using FORM + KI or FORM + PVP-I under irradiation [11].
