**2. Photosensitizer activation modes**

#### **2.1 Dark activity**

*Microorganisms*

susceptible to OH.

**1.3 Photosensitizers for PACT**

forgotten for several decades. However, interest in antibacterial PDT has been rekindled and is continuously increasing because multidrug resistance of pathogenic microorganisms has become a serious threat to public health. Photodynamic antibacterial chemotherapy (PACT) has become a promising approach for combat-

PACT is based on the exposure of bacteria to photosensitive compounds—photosensitizers (PSs). When a PS located in the bacteria or on the bacterial surface is exposed to light (usually visible), it transfers from its low-energy ground state to an excited singlet state. Return of the PS to its ground state is accompanied by either emission of fluorescence or transition of the PS to a longer-living, higherenergy triplet state (PS\*) via intersystem crossing. The PS\* in turn reacts with surrounding molecules to form free radicals and hydrogen peroxide (Type I reaction) or transfers its energy to molecular oxygen to produce singlet oxygen and other highly reactive oxygen species (ROS; Type II reaction) [9, 10]. Type I and Type II reactions occur simultaneously, and the ratio at which they occur depends on both the PS type and the surrounding conditions. A detailed description of the photosensitization process can be found in the recent reviews of Castano et al. [11] and Cieplik [10]. ROSs formed in this process oxidize biomolecules, damage the cell membrane, and ultimately lead to cell death [12]. PACT usually proceeds predominantly through Type II processes. However, since Gram-negative bacteria are more

radicals than to singlet oxygen, the Type I reaction may be more

Hundreds of compounds are currently available for mediating PDT in various areas of medicine, where some have been shown to be suitable for antimicrobial applications. PSs employed for medical uses should be a single pure compound, stable at room temperature and inexpensive. The PS must have a strong absorption peak in the visible spectrum between 600 and 900 nm and should possess a hightriplet quantum yield that will provide high production of ROS upon illumination. It should not be toxic in the dark (especially to mammalian cells), mutagenic or carcinogenic [15–18]. In addition, when talking about PACT, it is very important that the PS will display preferential association with bacteria, accumulate within

PSs can generally be assigned to several chemical classes: tetrapyrroles (which include porphyrins, chlorins, bacteriochlorins, and phthalocyanines), synthetic dyes (phenothiazinium salts, Rose Bengal, squaraines, etc.), and naturally occurring compounds (such as riboflavin or curcumin). Cyclic tetrapyrroles present the most well-known class of clinically relevant PSs used mostly for anticancer applications [20]. This structure can be found naturally in such important biomolecules such as haem, chlorophyll, and bacteriochlorophyll. Unlike other types of PSs, most tetrapyrroles (except for bacteriochlorins) are more likely to react by a Type II reaction with the creation of singlet oxygen [16], whereas bacteriochlorins act *via* a Type I mechanism. Other well-known antimicrobial agents are phenothiaziniumbased synthetic dyes, including methylene blue (MB) and toluidine blue O (TBO), which also act as anticancer agents in PDT. These structures can be synthesized more easily than tetrapyrroles but possess high-dark toxicity compared to other PSs [15, 21]. Another representative of synthetic dyes, Rose Bengal (RB), has already been used successfully in antimicrobial and anticancer applications for a long

ing bacterial infections, which are resistant to modern antibiotics.

**1.2 Photosensitizers and their mechanism of action**

efficient against such microorganisms [13, 14].

the cells, or bind to the bacterial cell envelope [14, 19].

**132**

The name photosensitizer implies the need for illumination in order to activate PS molecules and trigger their action. However, PSs possess some so-called "dark activity" even in the absence of illumination, leading to cell death in the dark [23–29]. This feature depends on the PS concentration and manifests itself in different ways for various PSs.

Shrestha demonstrated dark toxicity of RB against Gram-positive *Enterococcus faecalis*. Exposure of the cells to 10 μM RB in the absence of illumination for 15 min led to a 0.5 log10 reduction in cell concentration [26]. Furthermore, a marked dark toxicity of RB against clinical isolates of Gram-negative *Pseudomonas aeruginosa* was observed by Nakonieczna [27]. Brovko compared the activity of various PSs against several types of microorganisms and noted high dark toxicity of RB, as well as of phloxine B against Gram-positive *Bacillus sp*. and *Listeria monocytogenes* (more than 5 log10 reduction in the bacterial concentration after 30 min of treatment with the dye) [30]. The toxicity of malachite green in the dark against the same microorganisms was very low (<0.1 log10 reduction in concentration after 30 min of treatment with the dye). High concentrations (>500 μg/mL) of acriflavin neutral in the absence of light were significantly toxic to *E. coli* (more than 6 log10 reduction in concentration after 30 min of treatment with the dye, both under illumination and

#### **Figure 1.**

*Effect of RB concentration on its cytotoxic activity. S. aureus cells at the initial concentration of 104 CFU mL−1 were incubated for 3 min in dark conditions at various concentrations of RB. After the incubation, bacteria were tested by viable count. Error bars present standard deviations.*

in the dark). However, illumination significantly enhanced its toxic effect against other tested microorganisms [30].

In our studies, we also noted the dark toxicity of various PSs against different types of bacteria (**Figures 1, 2**, **Table 1**). **Figure 1** shows the effect of various RB concentrations on *S. aureus* in the absence of light. The number of living cells decreases with increasing RB concentration in the dark. **Table 1** shows a comparison between dark and light toxicity of three PSs—malachite green oxalate (MGO), RB, and safranin O. The effect of MGO in the dark was the strongest, and a 0.87 μM concentration of MGO was sufficient for inhibiting the growth of *S. aureus*. The dark activity of RB and safranin O is noticeably weaker, and the minimal inhibitory

#### **Figure 2.**

*SACT and PACT effect of MB on S. aureus. In SACT experiments, the cells at 108 CFU mL<sup>−</sup><sup>1</sup> concentration were incubated with (a) 5 μM RB or (b) 30 μM MB in the ultrasonic bath for 1 h in the dark. In PACT experiments, the cells were illuminated for 15 min by 1.6 mW cm<sup>−</sup><sup>2</sup> white light under the same conditions but without sonication. After the treatment, bacteria were tested by viable count. Error bars present standard deviations.*

**135**

*Aspects of Photodynamic Inactivation of Bacteria DOI: http://dx.doi.org/10.5772/intechopen.89523*

*at room temperature by white light of 1.6 mW cm<sup>−</sup><sup>2</sup>*

**2.2 Illumination**

**Table 1.**

*shaking at 37°C.*

for Safranin O.

concentrations (MIC) for these PSs against *S. aureus* are more than 100-fold higher. **Figure 2** shows that *S. aureus* cells were completely destroyed by RB at a concentration of 5 μM and MB at 30 μM under illumination. These PSs also showed a cytotoxic effect when applied at the same concentrations in the dark, where MB reduced

aureus *were treated by malachite green oxalate, Rose Bengal, and Safranin O at doubled dilutions, illuminated* 

**Dark Illumination**

 *intensity for 1 h, and incubated overnight in the dark by* 

 *of* S.

Although PSs are known to possess a certain dark activity, illumination noticeably increases their cytotoxic effect [6, 14]. An example of the difference in antibacterial activity of different PSs with and without illumination is shown in **Table 1**. In this experiment, the MIC of three PSs was determined for the bacterium *S. aureus* in the dark and after 1 h of illumination. As a result of illumination, the MIC of the examined PSs decreased approximately 6-fold for MGO, 64-fold for RB, and 4-fold

The main light sources used today for activation of PSs are lasers, light-emitting diodes (LED), and gas discharge lamps (GDL) [10, 31, 32]. There is no absolute advantage of one of these light sources over the others. The choice of light source depends on the specific application. Laser is a high-intensity monochromatic source. It can be easily coupled to a single optical fiber and installed on different lighting devices. LED lamps are cheaper and provide a wide emission spectrum. GDLs are also cheaper than lasers—both in acquisition and in maintenance and have a wide emission spectrum. However, GDLs transmit more heat to the illuminated area than lasers and LEDs, which can lead to tissue damage. In general, the emission spectrum and light intensity are more important for the excitation of a

Illumination is undoubtedly the easiest and most effective way to activate PSs. However, its use is restricted, due to limited penetration of visible light into tissues. There is an ongoing search for alternative methods of PS excitation in the dark in order to overcome this problem. Ultrasonic activation seems to be attractive as an alternative to illumination. As with light activation, ultrasound can be selectively focused on a specific area, thus activating only PS molecules located in the affected area. Ultrasound can also easily penetrate into tissues, which opens prospects for its application in treatment of internal lesions and infections, without the need for invasive devices [33, 34]. Ultrasonic irradiation of PSs initiates the formation of highly active cytotoxic species—ROS and free radicals—which lead to the death of pathogenic cells. It was found that some well-known PSs also have sonosensitizing properties. Among

the bacterial concentration by one and RB by two orders of magnitude.

**Photosensitizer MIC, μM**

*The MIC values of water-soluble PSs in the dark and under illumination. About 3 × 104 CFU mL<sup>−</sup><sup>1</sup>*

Malachite green oxalate 0.87 0.15 Rose Bengal 128 2 Safranin O 89 23

specific PS than the particular light source type [10, 31, 32].

**2.3 Sonodynamic excitation of photosensitizers**


**Table 1.**

*Microorganisms*

other tested microorganisms [30].

in the dark). However, illumination significantly enhanced its toxic effect against

In our studies, we also noted the dark toxicity of various PSs against different types of bacteria (**Figures 1, 2**, **Table 1**). **Figure 1** shows the effect of various RB concentrations on *S. aureus* in the absence of light. The number of living cells decreases with increasing RB concentration in the dark. **Table 1** shows a comparison between dark and light toxicity of three PSs—malachite green oxalate (MGO), RB, and safranin O. The effect of MGO in the dark was the strongest, and a 0.87 μM concentration of MGO was sufficient for inhibiting the growth of *S. aureus*. The dark activity of RB and safranin O is noticeably weaker, and the minimal inhibitory

**134**

**Figure 2.**

*deviations.*

*SACT and PACT effect of MB on S. aureus. In SACT experiments, the cells at 108 CFU mL<sup>−</sup><sup>1</sup>*

*experiments, the cells were illuminated for 15 min by 1.6 mW cm<sup>−</sup><sup>2</sup>*

*were incubated with (a) 5 μM RB or (b) 30 μM MB in the ultrasonic bath for 1 h in the dark. In PACT* 

*without sonication. After the treatment, bacteria were tested by viable count. Error bars present standard* 

 *concentration* 

 *white light under the same conditions but* 

*The MIC values of water-soluble PSs in the dark and under illumination. About 3 × 104 CFU mL<sup>−</sup><sup>1</sup> of* S. aureus *were treated by malachite green oxalate, Rose Bengal, and Safranin O at doubled dilutions, illuminated at room temperature by white light of 1.6 mW cm<sup>−</sup><sup>2</sup> intensity for 1 h, and incubated overnight in the dark by shaking at 37°C.*

concentrations (MIC) for these PSs against *S. aureus* are more than 100-fold higher. **Figure 2** shows that *S. aureus* cells were completely destroyed by RB at a concentration of 5 μM and MB at 30 μM under illumination. These PSs also showed a cytotoxic effect when applied at the same concentrations in the dark, where MB reduced the bacterial concentration by one and RB by two orders of magnitude.
