Rhamnolipid exhibits anti-biofilm activity against the dermatophytic fungi Trichophyton rubrum and Trichophyton mentagrophytes

Abstract

Dermatophytes are responsible for a majority of fungal infections in humans and other vertebrates, causing dermatophytosis. Treatment failures are often associated with biofilm formation, making dermatophytes resistant to antifungals. In this study, effects of a rhamnolipid (RL-SS14) produced by Pseudomonas aeruginosa SS14 on planktonic cells of Trichophyton rubrum and Trichophyton mentagrophytes, their biofilm formation, and disruption of mature biofilms were assessed. The composition of RL-SS14 was analysed using FTIR, HPLC-ESI-MS, and GC-MS. Minimum inhibitory concentrations against the planktonic forms of T. rubrum and T. mentagrophytes were 0.5 mg/mL and 0.125 mg/mL, respectively. Crystal-violet (biofilm biomass) and safranin (extracellular matrix) staining revealed that RL-SS14 significantly inhibited biofilm formation and also reduced preformed biofilms in a dose-dependent manner. Microscopic visualization of treated biofilms via SEM, AFM, and CLSM revealed marked morphological damage, cell death, and reduced extracellular matrix. The results indicate the potential of RL-SS14 as an anti-biofilm agent against dermatophytes.

Keywords: Anti-biofilm activity; Biosurfactant; Rhamnolipids; Trichophyton spp.; Ultramicroscopy.

Fig. 1

Inhibition of biofilm formation of Trichophyton rubrum and Trichophyton mentagrophytes on treatment with a rhamnolipid produced by Pseudomonas aeruginosa SS14 (RL-SS14) as determined by crystal violet (CV) and safranin staining. Commercial rhamnolipid (R-95) and terbinafine (TBF) were used as reference standards. Results are mean ± standard deviation (SD) and obtained from three independent experiments. Three replicate wells per condition were used. Different letters within each concentration indicate significantly different values as per the least significant difference (LSD) test.

Fig. 2

Effect of rhamnolipid produced by Pseudomonas aeruginosa SS14 (RL-SS14) on pre-formed biofilms of Trichophyton rubrum and Trichophyton mentagrophytes as determined by crystal violet (CV) and safranin staining. Commercial rhamnolipid (R-95) and antifungal terbinafine (TBF) were used as reference standards. Results are mean ± standard deviation (SD) and obtained from three independent experiments. Three replicate wells per condition were used. Different letters within each concentration indicate significantly different values as per the least significant difference (LSD) test.

Fig. 3

Representative scanning electron microscopic (SEM) images of the effect of rhamnolipid produced by Pseudomonas aeruginosa SS14 (RL-SS14) and Terbinafine (TBF) on mature biofilms of Trichophyton rubrum and Trichophyton mentagrophytes. The samples were treated at 2 × MIC of RL-SS14 (1 mg/mL for T. rubrum and 0.25 mg/mL for T. mentagrophytes) or TBF (0.03 mg/mL for T. rubrum and 0.062 mg/mL for T. mentagrophytes). The morphological changes were visualized and compared to the untreated control samples. Scale bar is 20 μm (2000×) and 2 μm (5000×).

Fig. 4

Representative two and three-dimensional atomic force microscopic (AFM) images of the effect of rhamnolipid produced by Pseudomonas aeruginosa SS14 (RL-SS14) on mature biofilms of Trichophyton rubrum and Trichophyton mentagrophytes. Samples treated with terbinafine (TBF) served as the standard drug control. The topographical changes in the biofilms induced on treatment with 2 × MIC of RL-SS14 (1 mg/mL for T. rubrum and 0.25 mg/mL for T. mentagrophytes) and TBF (0.03 mg/mL for T. rubrum and 0.062 mg/mL for T. mentagrophytes) were visualized and compared to the untreated control samples.

Fig. 5

Representative images obtained after confocal laser scanning microscopy (CLSM) of the biofilms of Trichophyton rubrum and Trichophyton mentagrophytes stained with propidium iodide (PI) after exposure to 2 × MIC of a rhamnolipid produced by Pseudomonas aeruginosa SS14 (RL-SS14; 1 mg/mL for T. rubrum and 0.25 mg/mL for T. mentagrophytes) or Terbinafine (TBF; 0.03 mg/mL for T. rubrum and 0.062 mg/mL for T. mentagrophytes). Untreated samples served as a negative control. Scale bar is 25 μm.