*2.3. Biological Activities*

2.3.1. Antibacterial and Anti-*Candida Albicans* Activities

The three EPS fractions were screened for their ability to inhibit the growth of four bacterial strains (*Staphylococcus aureus* ATCC 29213, methicillin resistant *Staphylococcus aureus* (SMR), *Escherichia coli* ATCC 25922 and *Salmonella* Enteritidis ATCC 13076) and the yeas<sup>t</sup> *Candida albicans* ATCC 10231.

All 3 EPS samples showed an antibacterial activity against the different bacterial strains tested. However, no antifungal activity against *Candida albicans* was detected. The most active was the native EPS that exhibited antibacterial activity against *E. coli* and *Salmonella* (Gram (−)) at MIC (Minimum Inhibitory Concentration) of 62.5 μg/mL and 125 μg/mL, respectively. This extract has also shown growth inhibition of Gram (+) bacteria at MIC 125 μg/mL for *S. aureus* and 1000 μg/mL for SMR. EPS-2C inhibited Gram (−) bacteria at a concentration of 2500 μg/mL and exhibited activity against *S. aureus* at a MIC of 1250 μg/mL. Finally, EPS-5C showed an antibacterial activity against *E. coli* and *S. aureus* at a concentration of 2500 μg/mL (Table 3). The MIC value of the different EPS samples was higher than that of the reference antibiotics Cefazolin and Amphotericin B that were used as positive controls. Even if the antimicrobial potential of these conventional antimicrobial agents was higher than that of EPS, the later could represent a natural and safer molecule without secondary effects.



MIC: Minimal Inhibitory Concentration. (-): Not determined. Cefazolin and Amphotericin B were used as positive controls.

> These antibacterial activities were also demonstrated by the appearance of an inhibition zone on agar medium using the well diffusion method at MIC concentrations obtained previously (Figure 3). According to this test, it clearly appeared that the various exopolysaccharide fractions have an inhibitory effect on the growth of the bacteria, manifested by the formation of an inhibition zone around the wells. This area depended on the sensitivity of the bacterial strain to the EPS extract and its concentration. Indeed, *Escherichia coli* ATCC 25922 was found to be the most sensitive Gram (−) bacterium to EPS-0C with a MIC of 62.5 μg/mL while *Staphylococcus aureus* ATCC 29213 was the most sensitive Gram (+) bacterium to EPS-0C with a MIC equal to 125 μg/mL. This sensitivity was revealed by an inhibition zone which was the most extensive in comparison with those of the other polysaccharide extracts (Figure 3), which were active only at concentrations greater than or equal to 1000 μg/mL. Thus, the native EPS (EPS-0C) seemed to be the most active compared to other exopolysaccharide extracts (EPS-2C and EPS-5C).

**Figure 3.** Zones of inhibition of the different bacterial strains affected by the exopolysaccharide extracts. (**A**): Activity against *E. coli*, 1: EPS-0C, 2: EPS-2C, 3: EPS-5C, 4: water, (**B**): Activity against *S. aureus*, 1: EPS-0C, 2: EPS -5C, 3: water, 4: EPS-2C, (**C**): Activity against *Salmonella*, 1: EPS-0C, 2: water, 3: EPS-2C, (**D**): Activity against SMR, 1: EPS-0C, 2: water.

#### 2.3.2. Inhibition of Biofilm Formation by *Candida Albicans* ATCC 10231

*P. marinum* EPS extracts were tested for their ability to inhibit *Candida albicans* biofilm formation by the 96-well plate crystal violet test. The three fractions (EPS-0C, EPS-2C and EPS-5C) were revealed to be active against the formation of *Candida albicans* biofilm (Figure 4) without affecting the planktonic growth of this yeast, as no mortality had been observed previously (Table 3).

**Figure 4.** Representative reverse microscopy images of the biofilm formed by *C. albicans* ATCC 10231 in absence (positive control: T+) or in the presence of exopolysaccharides at active concentrations. (**A**): EPS-0C at 31.3 μg/mL; (**B**): EPS-2C at 125 μg/mL and (**C**): EPS-5C at 250 μg/mL. The assay was performed on a 96-well plate for 24 h at 37 ◦C and the adherent biofilm cells were stained by crystal-violet.

Based on this observation, active EPS do not appear to target cell viability; they instead represent compounds whose mechanism of action would be the inhibition of the virulent form of *C. albicans*, in particular the transition from the yeas<sup>t</sup> form to the mycelial form. As shown on Figure 5, the native polymer EPS-0C was the most active compared to other fractions (EPS-2C and EPS-5C). In fact, it has shown an inhibition of 90 ± 1% at a concentration equal to 31.3 μg/mL, not significantly different from the efficiency of farnesol, used as a positive control, which inhibits 91% of *C. albicans* biofilm formation at 31.3 μg/mL concentration. However, EPS-2C inhibited the formation of biofilm by up to 78 ± 2% at 125 μg/mL and EPS-5C had an activity of 83 ± 3% at a concentration of 250 μg/mL.

**Figure 5.** Percentage of inhibition of *Candida albicans* biofilm formation by different molar masses EPS from *P. marinum* (EPS-0C, EPS-2C and EPS-5C) and at different concentrations between 7.8 and 250 μg/mL. Farnesol was used as a positive control. Results were expressed as mean ± standard deviations of triplicate determinations. Different lowercase letters stand for significantly different values (Duncan multiple range test, *p* <0.05).

#### 2.3.3. T1 Mammary Carcinoma Cells Sensitivity towards EPS Samples

The results of the in vitro test of the different EPS fractions on a murine breast carcinoma 4T1 cell lines have shown an antiproliferative activity on cancer cells mainly for EPS-2C and EPS-5C fractions (Figure 6). Indeed, the EPS-0C extract at the concentration of 2 mg/mL showed no significant inhibition of cancer cells with a percentage of viability equal to 85 ± 0.8%, which was found to not be significantly different from the viability obtained at the lower concentration (>90%). This result highlights the fact that the native EPS has no activity on carcinoma cells proliferation. In contrast, the antiproliferative activity was improved with the EPS-2C and EPS-5C exopolysaccharides having lower molar masses, as they were found to reduce the cell viability by 51 ± 0.75% and 45 ± 0.82%, respectively at a concentration of 2 mg/mL. As shown on Figure 6, EPS-2C and EPS-5C inhibited the proliferation of cancer cells in a concentration-dependent manner. In fact, the more the concentration (from 0.0625 to 2 mg/mL) increased, the more the antiproliferative activity of cancer cells increased, but with no statistically different behavior between the 2 samples.

Cytotoxicity of EPS samples was assayed on mammalian cell line Vero. Viability of cells was evaluated for each sample and at different concentrations (up to 5 mg/mL). Results showed that on the whole range of concentrations tested (between 9.75 to 5000 μg/mL), and for the 3 extracts (EPS-0C, EPS-2C and EPS-5C), no significant difference in viability was observed, and that it was almost 100% in all cases. As the CC50 (The 50% cytotoxic concentration, defined as the sample concentration able to reduce the cell viability by 50% when compared to an untreated control) of the samples were evaluated to be >5 mg/mL, and that no cytotoxic effect was observed for these samples in the range of concentrations used for the XTT test, the decrease in cell viability of carcinoma cells (about 50%) observed for 2 mg/mL concentrations could not be attributed to a cytotoxicity of the extracts and thus confirmed the antiproliferative effect.

**Figure 6.** Percentage of viability of murine mammary carcinoma cells 4T1 under the action of EPS samples from *P. marinum* (EPS-0C, EPS-2C and EPS-5C), at concentrations between 0.0625 and 2 mg/mL. Results were expressed as mean ± standard deviations of triplicate determinations. Different lowercase letters stand for significantly different values (Duncan multiple range test, *p* < 0.05).
