Evaluation of Antimicrobial Properties and Potential Applications of Pseudomonas gessardii M15 Rhamnolipids towards Multiresistant Staphylococcus aureus

Abstract

Staphylococcus aureus is a Gram-positive opportunistic human pathogen responsible for severe infections and thousands of deaths annually, mostly due to its multidrug-resistant (MDR) variants. The cell membrane has emerged as a promising new therapeutic target, and lipophilic molecules, such as biosurfactants, are currently being utilized. Herein, we evaluated the antimicrobial activity of a rhamnolipids mixture produced by the Antarctic marine bacterium Pseudomonas gessardii M15. We demonstrated that our mixture has bactericidal activity in the range of 12.5-50 µg/mL against a panel of clinical MDR isolates of S. aureus, and that the mixture eradicated the bacterial population in 30 min at MIC value, and in 5 min after doubling the concentration. We also tested abilities of RLs to interfere with biofilm at different stages and determined that RLs can penetrate biofilm and kill the bacteria at sub-MICs values. The mixture was then used to functionalize a cotton swab to evaluate the prevention of S. aureus proliferation. We showed that by using 8 µg of rhamnolipids per swab, the entire bacterial load is eradicated, and just 0.5 µg is sufficient to reduce the growth by 99.99%. Our results strongly indicate the possibility of using this mixture as an additive for wound dressings for chronic wounds.

Keywords: MRSA; S. aureus; SEM; biofilm; biosurfactants; rhamnolipids; wound dressing.

Figure 1

Biosurfactant overview (a); and mono-RL general structure (b).

Figure 2

Time-kill assay of M15RL against 6538. At the MIC concentration, M15RL was able to completely eradicate the bacterial load in 30 min. It requires just 5 min at 2 × MIC.

Figure 3

SEM visualization of S. aureus cells treated with M15RL in the microtiter assay conditions (a). In control (b) and in the sample treated with M15RL at concentration lower than MIC (c), S. aureus cells present a healthy morphology. Differently, when treated with MIC concentration (d) and over (e,f), cells lose their integrity, and cytoplasmatic material is reversed outside the cells. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated bacterium. **** p < 0.0001.

Figure 4

M15RL interference with the attachment of (a) S. aureus 6538 and (b) MRSA to the substrate. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated bacteria. **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, ns (not statistically significant).

Figure 5

Inhibition of mature biofilm formation against (a) S. aureus 6538 and (b) MRSA. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated bacteria. **** p < 0.0001, *** p < 0.0002, ns (not statistically significant).

Figure 6

The ability to kill S. aureus 6538 (a) and MRSA (b) bacteria inside the biofilm was evaluated by the biofilm penetration assay. Results are presented as the percentage reduction of metabolically active cells. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated biofilms. **** p < 0.0001, ns (not statistically significant).

Figure 7

Degradation of mature biofilms produced by (a) S. aureus 6538 and (b) MRSA. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated bacteria. **** p < 0.0001, ** p < 0.0021, * p < 0.0332, ns (not statistically significant).

Figure 8

Experimental design of the cotton swab assay (a), created with BioRender.com. M15RL-soaked swabs exhibited high antimicrobial activity (b).

Figure 9

The ability of M15RL to interfere with mammalian cells. Thus, the cytotoxicity was assessed by hemolysis assay. Statistical analyses were determined by ANOVA with Dunnett’s test for multiple comparisons. Significances are referred to the untreated red cells. **** p < 0.0001, ns (not statistically significant).

Figure 10

Stability of M15RL to high temperatures and influence of pH on the M15RL MIC. The mixture showed high resistance to high temperatures, being affected only after 40 min at 121 °C (a). On the other side, the antimicrobial activity of M15RL is highly affected by the pH of the environment, being less active at pH levels higher than 7 (b).