Seaweed Extracts: A Promising Source of Antibiofilm Agents with Distinct Mechanisms of Action against Pseudomonas aeruginosa

Abstract

The organization of bacteria in biofilms is one of the adaptive resistance mechanisms providing increased protection against conventional treatments. Thus, the search for new antibiofilm agents for medical purposes, especially of natural origin, is currently the object of much attention. The objective of the study presented here was to explore the potential of extracts derived from three seaweeds: the green Ulva lactuca, the brown Stypocaulon scoparium, and the red Pterocladiella capillacea, in terms of their antibiofilm activity against P. aeruginosa. After preparation of extracts by successive maceration in various solvents, their antibiofilm activity was evaluated on biofilm formation and on mature biofilms. Their inhibition and eradication abilities were determined using two complementary methods: crystal violet staining and quantification of adherent bacteria. The effect of active extracts on biofilm morphology was also investigated by epifluorescence microscopy. Results revealed a promising antibiofilm activity of two extracts (cyclohexane and ethyl acetate) derived from the green alga by exhibiting a distinct mechanism of action, which was supported by microscopic analyses. The ethyl acetate extract was further explored for its interaction with tobramycin and colistin. Interestingly, this extract showed a promising synergistic effect with tobramycin. First analyses of the chemical composition of extracts by GC-MS allowed for the identification of several molecules. Their implication in the interesting antibiofilm activity is discussed. These findings suggest the ability of the green alga U. lactuca to offer a promising source of bioactive candidates that could have both a preventive and a curative effect in the treatment of biofilms.

Keywords: Pseudomonas aeruginosa; Ulva lactuca; anti-biofilm; biofilm-matrix; seaweed extracts; synergistic activity.

Figure 1

Effect of different algal extracts (50.0 µg/mL) on PAO1 biofilm formation, assessed using the CV staining method. Extracts were added at t₀ to evaluate their effect on biofilm formation and growth. Results are expressed as the inhibition percentage (IP_CV %) mean ± SD, from three independent experiments. CH, DCM, EA, and MeOH are cyclohexane, dichloromethane, ethyl acetate, and methanol extracts, respectively. Statistically significant difference (**, p-value < 0.01, ***, p-value < 0.001) between the extract and the related untreated control is indicated.

Figure 2

Epifluorescence microscopy images of PAO1 biofilms incubated in MBB medium at 37 °C for 24 h without extract (control) or with one of the two active extracts (cyclohexane or ethyl acetate extract) of the green alga U. lactuca at 50.0 μg/mL. Extracts were added at t₀. Biofilms were stained with Syto9 for cells (green-fluorescent), with concanavalin A for the matrix sugars (red-fluorescent), and with Syto9 and propidium iodide (PI) to differentiate live and damaged cells, respectively. U.l (CH) and U.l (EA) are cyclohexane and ethyl acetate extract, respectively, derived from the green alga U. lactuca. (Magnification: ×20).

Figure 3

Effect of selected algal extracts (50.0 µg/mL) on PAO1 24 h-old biofilm assessed using the CV staining method. Extracts were added at t_24h to evaluate their effect on 24 h-old biofilms. Results are expressed as the eradication percentage mean ± SD from three independent experiments. Statistically significant difference (*, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001) between the extract and the related untreated control is indicated. ND: not determined.

Figure 4

Effect of the U. lactuca EA extract (50.0 μg/mL) on PAO1 24 h-formed biofilm using the CFU counting assay. Both adherent and planktonic bacteria were quantified (CFU counts) after 24 h incubation in the MBB medium. Results are expressed as mean (log CFU/mL) ± SD from three independent experiments. Statistically significant difference (**, p-value < 0.01) between extract and control is indicated. EA: ethyl acetate extract. NS: not significant.

Figure 5

Synergistic effect of the U. lactuca EA extract (50.0 µg/mL) and tobramycin (2 µg/mL) and colistin (16 µg/mL) on PAO1 biofilms using the CFU counting assay method. The EA extract was added at t₀. The EA extract/antibiotic combination was added after 24 h of incubation at 37 °C as antibiotics alone. Results are expressed as means of log reduction in comparison with the related untreated control (log reduction (log CFU/mL) ± SD) from three independent experiments. Statistically significant differences (**, p-value < 0.01, ***, p-value < 0.001) between the log CFU/mL number remaining after treatment with the EA extract/antibiotic combination or with the antibiotics alone and that in the appropriate untreated control are indicated. Statistically significant difference (*, p-value < 0.05) between the log CFU/mL number remaining after treatment with the EA extract/antibiotic combination vs. antibiotic alone. NS: not significant.